Chromatin Accessibility Thiab MicroRNA Qhia hauv Nephron Progenitor Cells Thaum Lub Raum Loj Hlob
Mar 11, 2022
Yog xav paub ntxiv:ali.ma@wecistanche.com
Andrew Clugston1,2,3, Andrew Bodnar2,3, Débora Malta Cerqueira2,3, Yu Leng Phua2,5,6Alyssa Lawler8,9,10, Kristy Boggs7, Andreas Pfenning8,10, Jacqueline Hlo2,3*thiab Dennis Kostka1,4*
1Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA2Rangos Research Center, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA3Department of Pediatrics, Division of Nephrology, University of Pittsburgh School of Medicine, PA, USA4Department of Computational & Systems Biology thiab Pittsburgh Center rau Evolutionary Biology thiab Tshuaj, University of Pittsburgh Tsev Kawm Ntawv ntawm Tshuaj, Pittsburgh, PA, USA5Division of Genetic and Genomic Medicine, Department of Pediatrics, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA6Department of Pathology, Clinical Biochemical Genetics Laboratory, UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA7Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.8Computational Biology,9Biological Sciences, thiab10Lub koom haum Neuroscience, Carnegie Mellon University, Pittsburgh, PA, USA
Abstract
Mammalian nephrons originates los ntawm cov pejxeemnephron progenitor hlwb, thiab kev hloov pauv ntawm cov cell 'transcriptomes ua rau muaj kev cuam tshuam ntawm nephrogenesis, qhov tseem ceeb ntawm tus lej nephron. Txhawm rau ua tus cwj pwm microRNA (miRNA) kev qhia thiab txheeb xyuas cov cheeb tsam cis-kev tswj hwm, peb tau sau nephron progenitor hlwb los ntawm nas.lub raumntawm embryonic hnub 14.5 thiab postnatal hnub xoom thiab soj ntsuam me me RNA qhia thiab transposase-accessible chromatin. Peb tau kuaj pom cov lus qhia ntawm 1,104 miRNA (114 nrog kev hloov pauv), thiab 46,374 chromatin nkag tau rau cheeb tsam (2,103 nrog kev hloov pauv tsis tau). Genome-wide, peb cov ntaub ntawv qhia txog cov txheej txheem xws li kev sib txawv ntawm tes, kev tsiv teb tsaws ntawm tes, kev sib cuam tshuam ntawm cov cellular matrix, thiab txoj kev loj hlob ntawm kev taw qhia. Tsis tas li ntawd, lawv txheeb xyuas cov neeg sib tw tshiab cis-kev tswj hwm cov ntsiab lus rau Eya1 thiab Pax8, ob lub noob nrog lub luag haujlwm hauv nephron progenitor cell sib txawv. Thaum kawg, peb koom nrog kev hloov pauv miRNAs, suav nrog cia-7-5p, miR-125b-5p, miR-181a-2-3p, thiab miR{{ 20}}p, nrog cov neeg sib tw cis-kev tswj hwm cov ntsiab lus thiab cov hom phiaj. Cov kev tshuaj ntsuam no qhia txog qhov tshiab putative cis-regulatory loci rau miRNA hauv nephron progenitors.
Taw qhia
Lub functional unit ntawm lubraumyog nephron, uas ua haujlwm lim dej pov tseg thiab tswj hwm homeostasis ntawm dej, acid-base, thiab electrolytes hauv lub cev. Nephron tus naj npawb sib txawv hauv tib neeg (feem ntau ntawm ob puas txhiab thiab ntau dua ib lab nephrons (Hughson li al., 2003)) thiab tsim ua ntej yug hauv tib neeg (kwv yees hnub tom qab yug menyuam 2-3 hauv nas (Hartman li al. ., 2007; Hinchliffe et al., 1991). Nephrons tsis tuaj yeem rov tsim dua tom qab yug me nyuam, thiab txo qis nephron endowment yog txuam nrog kev pheej hmoo ntawm cov kab mob raum ntev thiab kub siab (Bertram li al., 2011). Nephron tooj, nyob rau hauv lem, yog nyob rau hauv loj ib feem txiav txim los ntawm ib tug pejxeem ntawm lub hlwb hu uanephron progenitor hlwb(Cebrian et al., 2014b). Nyob rau theem kawg ntawmraumtxoj kev loj hlob, nephron progenitors nce lawv propensity rau txawv, uas maj depletes lawv cov pej xeem thiab cim qhov kawg cessation ntawm nephrogenesis (Hartman li al., 2007; Rumballe li al., 2011; Volovelsky li al., 2018). Kev nkag siab txog cov txheej txheem uas tswj kev txiav tawm ntawm nephrogenesis muaj qhov cuam tshuam loj rau kev txiav txim siab ntawm nephron endowment thiab kev pheej hmoo ntawm cov kab mob raum tom ntej.
Thaumraumtxoj kev loj hlob, nws tus kheej-renewing Cited1 plus /Six2 plus nephron progenitors yog induced los ua primed Cited1-/Six2 plus progenitors, thiab tom qab sib txawv rau Wnt4-qhia rau lub raum vesicles teb rau Wnt9b/ -catenin (McMahon thiab al., 2016; Rumballe et al., 2011). Intrinsic transcriptional hloov nyob rau hauv thaum ntxov piv rau lig nephron progenitors muaj xws li cov noob thiab txoj kev uas cuam tshuam qia cell laus, tshwj xeeb tshaj yog, mTor thiab nws repressor hamartin (S. Chen li al., 2015; Volovelsky li al., 2018). Tseeb tiag, hamartin yog xav kom desensitize nephron progenitors rau cov teeb liab rau nws tus kheej-renewal, thiab nyob rau hauv txoj kev no ua rau lub cessation ntawm nephrogenesis (S. Chen li al., 2015). Raws li lub tswv yim hais tias thaum ntxov nephron progenitors muaj kev nyiam ntau dua rau kev rov ua dua nws tus kheej yog qhov kev soj ntsuam uas chromatin nkag mus tau zoo rau kev khi qhov chaw ntawm cov tub ntxhais nephron progenitor cell transcription yam, suav nrog Six2 thiab Wt1 thaum piv rau nephron progenitors lig (Hilliard li al. , 2019). Cov kev hloov pauv no tau pom tias yuav khi cov kab ke txhim kho nyob rau hauv "kev tswj hwm kub-spots" hauv genome (O'Brien li al., 2018). Qhov induction ntawm nephron progenitors tshwm sim vim nce -catenin ntau ntau, uas ua rau kom ua rau Lef1 / Tcf7 complexes ntawm cov noob npaj rau kev ua kom lub nephrogenesis program (Park li al., 2012), thiab tau pom zoo kom complex nrog Tcf7l1 thiab Tcf7l2. los tsim thiab tswj kev tswj hwm chromatin loops uas pab txhawb kev ua kom cov noob (Guo li al., 2021).
Nyem rauCistanche tubulosa hmoov rau kev kho mob raum
Cov kev tshawb fawb tsis ntev los no tau cuam tshuam txog microRNAs (miRNA) hauv kev tswj cov kev txiav tawm ntawm nephrogenesis. miRNA yog luv luv, tsis-coding RNA molecules uas lub hom phiaj-RNA transcripts (mRNA) kom txo qis kev txhais lus lossis txhim kho degradation ntawm RNA-induced silencing complex (RISC) (Bartel, 2009). Cov protein Lin28b yog ib tug paub repressor ntawm let{5}}tsev neeg ntawm miRNAs (Heo et al., 2008). Lin28b kev qhia txo qis hauv nephron progenitors raws li nephrogenesis pib, thiab qhov no coincides nrog kev nce hauv kev qhia ntawm let-7 miRNA tsev neeg (Yermalovich li al., 2019). Tsis tas li ntawd, raws li txoj cai induction ntawm Lin28b qhia nyob rau hauv Wt1- kab mob tshwm sim nyob rau hauv kev tsuj ntawm cia-7 miRNA, uas yog txaus kom ntev nephrogenesis (Yermalovich li al., 2019). Nws tsis paub tias lwm tus miRNAs tswj lub sijhawm ntawm nephrogenesis. Tsis tas li ntawd, thaum lub sijhawm txhim kho uas tswj xyuas qhov ntev tsis-coding RNA qhia thaum lub sij hawm nephrogenesis tau txheeb xyuas (Nishikawa li al., 2019), tsis muaj leej twg tau pom tias tswj hwm miRNA kev qhia hauv nephron progenitors.
Hauv txoj kev tshawb no, peb tau tshawb nrhiav ib txhij txheeb xyuas cov tshiab miRNA uas ua rau muaj kev cuam tshuam ntawm nephrogenesis, nrog rau cov peev txheej cis-kev tswj hwm. Yog li ntawd, txhawm rau soj ntsuam cov kev hloov pauv hauv miRNA kev qhia thiab chromatin nkag tau ntawm embryonic hnub 14.5 (E14.5) thiab postnatal hnub xoom (P0), peb tsim mRNA-seq thiab ATAC-seq datasets los ntawm thawj thiab tsis dhau.nephron progenitor hlwbntawm ob lub sijhawm no. Peb tau txheeb xyuas 114 miRNAs uas tau qhia txawv hauvnephron progenitor hlwbnruab nrab ntawm ob lub sijhawm cov ntsiab lus no, nrog rau 2,103 thaj tsam ntawm kev hloov pauv chromatin nkag mus tau uas peb lub sijhawm sib txawv ntawm thaj chaw nkag tau (DARs). Enrichment analyses ntawm cov kwv yees lub hom phiaj ntawm kev hloov pauv loj miRNA nrog rau ntawm cov chaw genomic ntawm DARs cuam tshuam txoj hauv kev uas cuam tshuam rau kev sib txawv ntawm cov cell epithelial, cell migration, extracellular matrix kev sib cuam tshuam, thiab cov kev loj hlob tseem ceeb hauv kev taw qhia xws li Wnt, Notch, thiab TGF- signaling. . Peb pom cov kev hloov pauv tseem ceeb hauv kev nkag mus rau hauv chromatin thiab nthuav tawm ntau kab ntawm cov pov thawj cuam tshuam txog ob thaj chaw genomic ua cov tshuaj txhim kho rau cov noob Eya1 thiab Pax8 hauv nephron progenitor hlwb. Thaum kawg, peb tau txheeb xyuas qhov kev nkag mus tau zoo ib yam ntawm chromatin thiab miRNA kev qhia hloov pauv rau 33 khub ntawm miRNAs thiab DARs, suav nrog cia-7-5p, miR-125b-5p, miR-181a{ {11}}p, thiab miR-9-3p, thiab tam sim no lub hom phiaj cov noob tau nthuav tawm hauv nephron progenitors rau qhov sib txawv ntawm miRNAs.
Cov txiaj ntsig
Kev rho tawm ntawm nephron progenitor cell pejxeem los ntawm E14.5 thiab P0lub raum
Nephron progenitor hlwbraug cais los ntawm kev xaiv zoo rau Itg 8 ntawm E14.5 lossis ntawm P0, thiab txhua tus qauv tau muab faib ua mRNA-seq, ATAC-seq, thiab kev tswj kom zoo (Cov duab ntxiv 1 AB). Quantitative PCR (qRT-PCR) tau lees paub tias nephron progenitor marker genes Six2 thiab Cited1 (Huang et al., 2016) tau qhia ntau heev nyob rau hauv cais nephron progenitor qauv piv rau tag nrho.raum, ntau tshaj li cov cim rau lwm hom cell (xws li ureteric bud (Caleb) (Cebrian li al., 2{{10}}}14a), endothelial hlwb (Pecan) (Kobayashi li al., 2008), raum stroma (Pdgfr ) (S. Chen et al., 2015) thiab lub raum vesicle (Lhx1) (Brunskill li al., 2014)(Supplemental picture 1 C). Hais txog poj niam txiv neej, litter siv rau E14.5 qauv ranged los ntawm 27 feem pua mus rau 50 feem pua poj niam, thiab cov litters rau P0 cov qauv muaj li ntawm 31 feem pua rau 75 feem pua poj niam. Qhov nruab nrab, E14.5 cov qauv yog 38 feem pua poj niam, thiab P0 cov qauv yog 49 feem pua poj niam (Ntxiv daim duab 1 D). Cov no ua tib zoo hnub nyoog. -matched nephron progenitor cell pejxeem tso cai rau peb los soj ntsuamchromatin accessibilitythiab me RNA qhia nyob rau tib theem ntawm kev loj hlob ntawm ob lub sij hawm cov ntsiab lus (E14.5 thiab P0).
Thaum ntxov thiab lig nephron progenitors muaj qhov txawv ntawm miRNA transcriptional profiles
Me-RNA sequencing kuaj pom 1,104 paub miRNA transcripts thiab txheeb xyuas 114 miRNAs uas qhia tau hais tias muaj kev hloov pauv tseem ceeb ntawm E14.5 thiab P{{10}} cov qauv (kev tshawb pom cuav ( FDR)=0.05; Table 1). Ib nrab ntawm qhov sib txawv ntawm miRNAs qhia tau hais tias muaj kev nthuav dav ntxiv ntawm P0 piv nrog E14.5, thiab cov ntsiab lus tseem ceeb ntawm kev tsom xam qhia txog kev sib txawv ntau dua hauv miRNA kev qhia ntawm E14.5 cov qauv piv nrog P0 (Daim duab 1A). Hierarchical clustering qhia ib pawg meej ntawm miRNAs rau hauv cov kev nthuav qhia thiab txo qis ntawm E14.5 thiab P0, raws li (Daim duab 1B). Cov tswv cuab ntawm cia-7 tsev neeg ntawm miRNAs yog ib qho ntawm cov miRNA uas tau pom ntau tshaj plaws, thiab peb nco ntsoov tias txhua tus neeg hauv tsev neeg uas muaj kev hloov pauv tseem ceeb qhia tau hais tias ntau ntxiv ntawm P0 (cov ntaub ntawv tsis qhia). Qhov no yog nyob rau hauv txoj kab nrog cov txiaj ntsig uas qhia tau tias qhov txo qis hauv Lin28b qhia ua rau muaj kev nthuav dav dav hauv kev cia-7 tsev neeg qhia nyob rau hauv chav kawm ntawm nephrogenesis (Yermalovich li al., 2019). Lwm cov miRNAs uas muaj kev qhia ntau dua ntawm P0 suav nrog miR-429-3p, tus tswv cuab ntawm miR-200 tsev neeg paub tias cuam tshuam rau kev sib txawv ntawm podocyte (Z. Li li al., 2016), thiab ob qho tib si miR{{30 }}a-5p and miR-125b-5p. Ob qhov tom kawg no tau paub tias yuav txwv Lin28 cov ntawv sau tseg hauv cov chaw sib txawv ntawm tes (Chaudhuri li al., 2012; Potenza et al., 2017). miRNA nrog kev qhia qis qis ntawm P0 suav nrog cov angiogenesis- (S. Wang et al., 2008) thiab kev txhawb nqa kev loj hlob (Schober et al., 2014) miR-126.
Lub cuab yeej DIANA miRPath ua rau KEGG txoj kev tsom xam ntawm miRNA raws li lawv cov hom phiaj ntawm cov noob, raws li kev kwv yees los ntawm micro T-CDS (Vlachos li al., 2015). Peb pom tias miRNA nrog cov kev hloov pauv loj tshaj plaws ntawm E14.5 thiab P0 (nce lossis nqis) yog cov uas tswj txoj hauv kev nrog lub luag haujlwm tseem ceeb hauv nephrogenesis (Daim duab 1C, Daim duab ntxiv 2). Piv txwv li, Tgf- yog secreted los ntawm ib puag ncig stromal hlwb los txhawb nephron progenitor sib txawv (Rowan li al., 2{{50}}}18), thiab peb nco ntsoov txo cov kev qhia ntawm nephron progenitor miRNA tsom rau downstream effectors ntawm lub "TGF- signaling pathway" (KEGG pathway mmu04350, p-value 1.45e- 6). "MAPK signaling pathway" (mmu04010) cuam tshuam rau lub koom haum thiab priming ntawm nephron progenitors rau kev sib txawv, thiab nws tswj lawv cov kev cuam tshuam nrog extracellular matrix (ECM) ntawm Itg 8 (Ihermann-Hella li al., 2018). Peb tau sau tseg ib qho tseem ceeb ntawm cov miRNAs nce-thiab-tso-tswj cov miRNAs uas tsom cov noob sib txawv hauv MAPK qhov taw qhia txoj hauv kev (p-tus nqi ntawm 3.5e- 6 thiab 3.0e- 4 rau cov npe nce thiab nqis, feem) . Thaum kawg, KEGG txoj hauv kev "Extracellular matrix (ECM) receptor kev sib cuam tshuam" (mmu04512) yog txoj hauv kev uas muaj txiaj ntsig zoo tshaj plaws tau txheeb xyuas, thiab nws cov noob caj noob ces yog qhov tseem ceeb tshaj plaws los ntawm miRNA uas nce hauv kev qhia lub sijhawm (p-tus nqi ntawm 7.4e{ {32}} ntawm kev nce miRNA piv rau 1.1e-2 ntawm kev txo qis). ECM cov noob caj noob ces yog tsom los ntawm qee qhov kev tshaj tawm thiab nthuav dav miRNA peb pom, suav nrog miR-196a-5p, miR-148a-3p, miR -125a-5p, thiab cia-7f-5p. Tshwj xeeb, miR-196a-5p yog qhov siab tshaj plaws qhia miRNA hauv peb cov qauv, thiab nws pom qhov nce 8-fold nce ntawm E14.5 thiab P0. Gene transcripts uas yog lub hom phiaj kwv yees ntawm miRNA (siv TargetScan (Agarwal et al., 2015)) nrog kev nthuav qhia ntau ntxiv ntawm P0 suav nrog Bach2, ib qho kev sib txuas molecular ntawm MAPK / AP1 thiab Six2 / -catenin txoj hauv kev rau kev rov ua dua tus kheej thiab sib txawv. hauv nephron progenitors, ntsig txog (Hilliard li al., 2019).
Txhawm rau txheeb xyuas lub hom phiaj ntawm qhov sib txawv ntawm miRNAs hauv nephron progenitors, peb siv cov lus piav qhia ntawm miRNAs muab hauv miRDB (Y. Chen & Wang, 2020), thiab cov lus piav qhia kev sim miRNA-mRNA kev sib cuam tshuam los ntawm mortar puag (HY Huang li al., 2020), ua ke nrog peb yav dhau los tsim E14.5 ib leeg-cell RNA sequencing dataset (Bais li al., 2020). Yog li, peb tau tsim cov npe ntawm qhov muaj peev xwm miRNA-mRNA kev sib cuam tshuam raws li kev qhia sib txawv ntawm miR ntawm E14.5 thiab P0 hauv nephron progenitors nrog kev hloov pauv hauv cov hom phiaj ntawm cov noob qhia ntawm tus kheej-renewing thiab primed nephron progenitors, nrog rau primed thiab sib txawv nephron progenitors (Daim duab 2). Peb qhov kev soj ntsuam pom tau tias Meis2 thiab Hmga2 yog lub hom phiaj muaj peev xwm ntawm let{15}} tsev neeg ntawm miRNAs hauv nephron progenitors (Daim duab 2), nrog kev txo qis ntawm Meis2 thiab Hmga2 cuam tshuam nrog kev nthuav qhia ntau ntxiv ntawm let{19}} cov neeg hauv tsev neeg hauv kev sib txawv lossis kev laus nephron progenitors. Qhov kev hloov pauv ntawm Meis2 tau hais tawm muaj zog hauv nephron progenitors thiab lub raum stroma thaum lub sijhawmraumtxoj kev loj hlob (Lam et al., 2014), thiab txuas nrog kev tswj hwm ntawm kev loj hlob ntawm tes hauv lwm cov ntsiab lus (Abruzzese li al., 2019). Hmga2 yog nonhistone chromatin-binding protein, nws tau nthuav tawm heev hauv cov qia hlwb, thiab nws tau cuam tshuam nrog ntau yam ntawm cov qog nqaij hlav mesenchymal (Fusco & Fedele, 2007; Lam et al., 2014). Ib yam li ntawd, muaj kev nthuav qhia Jag1 cuam tshuam nrog kev txo qis ntawm miR-34b-5p, miR-983p, thiab miR-200a-3p hauv kev sib txawv los yog laus nephron progenitors (Daim duab 2). Jag1 yog ib qho tseem ceeb ligand hauv Notch signaling uas nws qhia tau nce raws li nephron progenitors txawv (Chung li al., 2016). Txhua qhov kev kwv yees lossis sau tseg miRNA lub hom phiaj rau cov noob nrog kev qhia hloov pauv ntawm tus kheej rov ua dua tshiab, primed, thiab sib txawv nephron progenitors muaj nyob rau hauv Ntxiv.
Daim duab 1. Cov kev hloov pauv loj hauv miRNA kev qhia ntawm E14.5 thiab P0 nephron progenitors.A) Kev tshuaj xyuas tus thawj tswj hwm qhia tau hais tias kev loj hlob lub sij hawm taw tes yog qhov tseem ceeb rau kev hloov pauv ntawm miRNA. B) Heatmap piav qhia txog cov txheeb ze ntawm txhua qhov hloov pauv miRNA. Cov kab uas muaj E14.5 cov qauv ntawm sab laug yog sau rau hauv lub teeb ntsuab, thiab kab nrog P0 cov qauv ntawm sab xis yog sau rau hauv ntsuab tsaus. C) Txhim kho cov noob los ntawm txoj hauv kev tshwj xeeb KEGG ntawm kev kwv yees cov hom phiaj ntawm nce-thiab qis-tswj miRNA (liab thiab xiav tuav, feem). Qhov tsawg kawg nkaus p-tus nqi suav nrog yog 1e-5.
Daim duab 2. miRNA-mRNA cuam tshuam nrog lub luag haujlwm ua tau hauv nephron progenitor cell aging thiab differentiation.Displayed is miRNA (circles) thiab mRNA (squares). miRNAs nrog kev nthuav qhia ntxiv ntawm E14.5 thiab P0 tau pom hauv liab, miRNAs uas txo qis yog pom hauv xiav. Daim ntawv teev cov hom noob caj noob ces (los ntawm mirDB thiab mir TarBase) nrog kev nthuav qhia ntxiv ntawm tus kheej-renewing, primed los yog txawvnephron progenitor hlwb(Bais li al., 2020) tau qhia hauv liab, mRNA nrog txo qis hauv xiav. Ntug los ntawm mirDB thiab mirTarDB uas tsis sib haum nrog kev pom kev hloov pauv tau raug tshem tawm tab sis suav nrog hauv Cov Lus Ntxiv 2.
Thaum ntxov thiab lig nephron progenitors muaj qhov sib txawv chromatin ib puag ncig
ATAC-seq cov ntaub ntawv los ntawm E14.5 thiab P0 nephron progenitors tso cai rau peb txheeb xyuas 46,374 thaj tsam ntawm chromatin nkag tau (nrog rau qhov kev tshawb pom tsis txaus ntseeg (Boleu li al., 2015) qhov pib ntawm {{ . B. FANTOM5 (de Rie li al., 2017) txhim kho cov ntaub ntawv, thiab 13,495 muaj DNA ib ntus nrog kev kwv yees kev tswj hwm hauv tsawg kawg ib tus nas cov ntaub so ntswg lossis hom cell raws li EnhancerAtlas 2.0 (Gao & Qian, 2020).
Kev tsom xam cov ntsiab lus tseem ceeb (hloov rau qhov sib txawv ntawm kev sib deev ntawm cov qauv) qhia txog kev qhia meej ntawm cov qauv los ntawm kev loj hlob lub sij hawm taw tes (Daim duab 3A). Txhawm rau txheeb xyuas genomic loci nrog kev hloov pauv hauv chromatin nkag tau, peb muab piv rau ATAC-seq teeb liab ntawm E14.5 thiab P0 cov qauv. Peb pom 2,103 DARs, tswj FDR ntawm 10 feem pua. Ntawm cov 1,323 (55 feem pua) tau pom tias muaj kev nkag tau ntau dua ntawm P0 piv nrog E14.5, thiab kev sib koom ua ke hierarchical qhia qhov txawv ntawm DARs uas qhib thiab kaw ntawm E14.5 thiab P0, raws li (Daim duab 3B).
Cov kev hloov pauv cov hneev taw tau txheeb xyuas siv HINT thiab sib phim rau DNA khi cov proteins uas siv cov motifs los ntawm HOCOMOCO 11 database (Kulakovskiy li al., 2018). Ntawm 431 motifs kuaj, DARs muaj cov hneev taw rau ntau qhov kev hloov pauv ntawm cov proteins uas cuam tshuam nrog nephrogenesis qhia tau tias muaj kev nce ntxiv ntawm E14.5 thiab P0. Ntawm cov hneev taw nyob hauv DARs uas nce qhov kev nkag tau ntawm qhov nruab nrab, rau saum toj kawg nkaus yog cov kev hloov pauv cov hneev taw tau sib tw rau Pax8 ({{10}}.15), Six2 (0.14), Cebpd (0.13), Six4 (0.13), Jun (0.12), thiab Fos (0.11). Cov hneev taw piav qhia nyob rau hauv kaw DARs suav nrog cov ntsiab lus hloov pauv E2F2 thiab E2F4, uas paub tias cuam tshuam rau lub voj voog ntawm tes (Gaudet li al., 2011) thiab txhawb kev loj hlob ntawm tes (D. Wang et al., 2000) hauv lwm cov ntsiab lus ntawm tes (cov duab ntxiv 4).
Txhawm rau soj ntsuam cov ntsiab lus lom neeg ntawm kev qhib thiab kaw DARs, peb siv Genomic Regions Enrichment of Annotations Tool (GREAT) (McLean li al., 2010) los qhia txog cov noob caj noob ces nrog kev txhawb nqa ntawm cov cheeb tsam hloov chromatin. Nrog rau txoj hauv kev ntawd, peb txheeb xyuas Gene Ontology (GO) cov txheej txheem lom neeg sib txuas rau nephron progenitor txoj kev loj hlob: tshwj xeeb, cov txheej txheem tseem ceeb tshaj plaws ntawm kev qhib DARs yog "Kev Cai ntawm epithelial cell sib txawv" (GO: 0030856, Binomial Q-value {{3 }}.5e-5), thiab "Kev tswj hwm zoo ntawm cov kab mob epithelial cell sib txawv" (GO: 0030858, Binomial Q-tus nqi=5e-3) yog ib qho ntawm 10 qhov tseem ceeb tshaj plaws enriched ( Daim duab ntxiv 5). Ob qho tib si zoo ib yam nrog kev nce ntxiv ntawm nephron progenitors mus rau kev hloov pauv mesenchymal mus rau epithelial (MET) raws li lawv sib txawv (S. Chen li al., 2015). Lwm txoj hauv kev muaj txiaj ntsig zoo suav nrog "Kev tswj hwm zoo ntawm Notch Signaling txoj hauv kev" (GO: 0045747, Binomial Q-tus nqi=2e-5), uas zoo ib yam nrog lub luag haujlwm Notch signaling plays hauv kev txhawb nqa nephron progenitor sib txawv ( Chung et al., 2016). Lub 10,083 kaw DARs kuaj pom tsis tau qhia GO cov ntsiab lus tseem ceeb raws li GREAT cov txheej txheem ua ntej rau qhov tseem ceeb (saib daim duab ntxiv 5).
Daim duab 3. Qhov txawv ntawm chromatin accessibility characterize E14.5 vs. P0nephron progenitor hlwb.A) Cov ntsiab lus tseem ceeb ntawm kev soj ntsuam ntawm ATAC-seq cov qauv qhia txog cov ntsiab lus ntawm lub sij hawm loj hlob raws li qhov loj ntawm kev hloov pauv. B) Heatmap qhia qhov hloov pauv IDR thaj tsam thoob plaws ATAC-seq cov qauv. E14.5 cov qauv/kem yog annotated nyob rau hauv lub teeb ntsuab, thiab P0 cov qauv/columns yog annotated nyob rau hauv ntsuab tsaus. C) GO Cov Txheej Txheem Txheej Txheem Zoo rau cov cheeb tsam genomic uas qhib nruab nrab ntawm E14.5 thiab P0 raws li GREAT, qeb los ntawm binomial P-tus nqi, FDR Tsawg dua lossis sib npaug rau 0.001.
Tom ntej no, peb tshawb nrhiavchromatin accessibilitynyob rau hauv cov cheeb tsam ntawm cov genome uas muaj cov neeg txhawb nqa thiab txhim kho cov noob nrog cov luag haujlwm tshaj tawm hauv nephron progenitor cell sib txawv. Ntawm cov no, Six2 transcription yog qhov tseem ceeb rau kev tswj hwm ntau lub zog ntawmnephron progenitor hlwb(Tus kheej li al., 2006), thiab nyob rau hauv tag nrho cov qauv, peb tau pom cov chromatin siv tau nyob ib puag ncig nws cov neeg txhawb nqa thiab paub txog Six2 enhancer ~ 50kb upstream ntawm Six2 transcription start site (TSS) (O'Brien li al., 2018) (Daim duab 4A, B, xiav-shaded cheeb tsam). Peb kuj tseem nco txog thaj tsam ntawm cov chromatin nkag tau rau ntawm tus neeg txhawb nqa thiab luam tawm kev txhim kho rau cov protein ntau Gdnf (Daim duab 4D, E), uas yog zais los ntawm nephron progenitors los txhawb nqa ureteric bud branching (Sanchez li al., 1996; Vega li al., 1996. ). Qhov no enhancer yog nyob ~ 113kb upstream ntawm cov noob txhawb nqa thiab paub tias yuav teb rau Wnt signaling nyob rau hauv cov ntsiab lus ntawm kev tsim nephron progenitors (Park li al., 2012). Thaum tus txhawb nqa rau Gdnf tsis hloov pauv ntau lub sijhawm, cov lus piav qhia ua kom zoo dua ua rau muaj kev nkag tsis tau zoo. Peb kuj tau sau tseg cov cheeb tsam ntawm cov chromatin siv tau nyob ib puag ncig ib qho kev tshaj tawm ua kom muaj zog hauv nephron progenitors rau Eya1 nyob ~ 325kb sab sauv ntawm cov noob TSS (Park et al., 2012) (Cov duab ntxiv 6). Eya1 yog ib qho kev hloov pauv uas cuam tshuam nrog Six2 kom tswj tau nephron progenitor multipotency (J. Xu li al., 2014).
Peb soj ntsuam ntau DARs uas tsis sib tshooj uas paub txog kev tswj hwm tab sis suav nrog thaj chaw kwv yees tias yuav muaj kev ua kom muaj zog ntxiv rau cov noob tshwj xeeb hauv ntau hom nas ntawm tes raws li sau tseg hauv EnhancerAtlas 2.0 (Gao & Qian, 2020). Ntxiv rau qhov kev tshaj tawm ntawm Eya1 (Park et al., 2012) (Cov duab ntxiv 6), qhov kaw DAR hauv ib qho ntawm Eya1's introns (Daim duab 5A-B) suav nrog thaj tsam kwv yees tias yuav muaj kev ua kom zoo dua ntawm Eya1 noob hauv nas. neural progenitor thiab granulocyte-monocyte progenitor cells (Gao & Qian, 2020). Nyob rau hauv txoj kab nrog kev txo qis hauv kev ua kom zoo dua qub, peb tau pom yav dhau los tias kev qhia ntawm Eya1 tau txo qis hauv kev sib txawv ntawm nephron progenitors siv E14.5 scRNA-seq cov ntaub ntawv (Cov duab ntxiv 7) (Bais li al., 2020). Qhov no zoo ib yam nrog cov ntaub ntawv luam tawm ChiP-seq hauv thawj E16.5 nephron progenitors uas qhia tias tsis muaj cov cim ua kom muaj zog, H3K27ac, thiab tsis muaj kev khi los ntawm Ctnnb1, Lef1, Six2, thiab Tcf7 (Daim duab 5) (Guo li al. Ib., 2021). Qhov kev txhim kho muaj peev xwm no tseem muaj cov ntaub ntawv hloov pauv rau Hoxd10 nyob rau hauv nws lub ncov, ib qho kev hloov pauv uas yog qhov tseem ceeb rau kev sib txawv thiab kev koom ua ke ntawm nephron progenitors thaum lub sij hawm mesenchymal-to-epithelial transitions (MET) (Yallowitz li al., 2011).
DAR uas ua rau kom tsis muaj peev xwm nkag mus tau ntawm chromosome 24 overlaps thaj tsam chromatin nrog kev kwv yees ua kom muaj txiaj ntsig zoo rau kev hloov pauv Pax8 hauv cov hlwb los ntawm nas lub raum, plab hnyuv epithelia, thiab uterus (Gao & Qian, 2020). Pax8 yog ib nrab redundant nrog Pax2, tab sis tsawg kawg yog ib qho ntawm ob qhov kev hloov pauv no yuav tsum tau muaj nyob rau hauv nephron progenitors rauraumkev loj hlob (Bouchard et al., 2002; Narlis et al., 2007). Peb yav dhau los tau pom tias Pax8 kev nthuav qhia nce hauv kev sib txawv thiab sib txawv ntawm cov nephron progenitors (Ntxiv daim duab 7), thiab peb tau pom qhov nce ntxiv hauv kev nkag mus tau zoo hauv DAR uas suav nrog qhov kev tswj hwm no. Cov cheeb tsam no tseem cuam tshuam nrog cov cim ua kom muaj zog, H3K27ac, thiab khi ntawm Ctnnb1, Lef1, Six2, thiab Tcf7 hauv cov ntaub ntawv luam tawm ChiP-seq los ntawm thawj E16.5 nephron progenitors (Daim duab 5) (Guo et al., 2021). Peb soj ntsuam ib tug xov tooj ntawm transcription yam hneev taw uas poob nyob rau hauv lub DAR no lub siab ceev kev nkag tau mus rau ncov, suav nrog ob rau Sall1, ib tug nuclear yam uas paub tias yuav cuam tshuam rau nephrogenesis (Kanda li al., 2014), thiab xya qhov sib koom ua ke hneev taw rau MYC-sociated zinc ntiv tes. Protein (Maz), ib qho kev hloov pauv uas tau pom tias muaj kev cuam tshuam ntau npaum li cas ntawmraumkev loj hlob ntawm tib neeg thiab nas (Haller li al., 2018). Cov Lus Qhia Ntxiv 3 muaj cov npe tag nrho ntawm thaj chaw nkag tau peb pom nrog rau lawv cov kev txheeb xyuas qhov sib txawv ntawm kev nkag mus tau zoo, cov lus piav qhia rau cov noob, exons, thiab cov neeg txhawb nqa uas lawv sib tshooj, thiab cov npe ntawm cov ntsiab lus tseem ceeb nephrogenesis-txuas kev sib txuas cov hneev taw taw qhia.
Daim duab 4. ATAC-seq profiles ntawm qhov paub thiab tso cai tswj qhov chaw.Rau txhua lub vaj huam sib luag los ntawm sab saum toj mus rau hauv qab, facets denote fold enrichment ntawmchromatin accessibilitynyob rau hauv E14.5 thiab P0 cov qauv, nrog IDRs thiab DARs txheeb xyuas qhov tseem ceeb hauv xiav (tsis hloov pauv IDR) thiab ntsuab (qhib DAR). thiab liab (kaw DAR). Cov nram qab no facet qhia txog cov noob annotations, nucleosomal txoj hauj lwm (grey) thiab nucleosome-dawb cheeb tsam (liab) ntsuas nyob rau hauv pooled E14.5 thiab P0 cov qauv, thiab PhastCon60 cov qhab nias kev txuag (dub qhia 1{{ 12}}0% kev txuag, dawb 0 feem pua kev txuag. B) Enhancer rau Six2 luam tawm los ntawm Park et. al. C) Sib txawv nkag mus tau zoo rau Gdnf nrog nce kev nkag tau ntawm P0. D) Txhawb nqa rau Gdnf.
Daim duab 5. DARs overlapping tej zaum tshiab enhancers rau nephrogenesis-txuas genes.A thiab C) Vaj huam sib luag piav qhia ib daim ntawv qhia ntawm 1MB cheeb tsam nyob ib ncig ntawm ib tug gene ntawm kev txaus siab, nrog protein-coding noob nyob rau hauv ntsuab thiab lub proposed enhancer/promoter kev sib cuam tshuam raws li ib tug dub voj. . B thiab D) Los ntawm sab saum toj mus rau hauv qab, panels depict fold enrichment ntawmchromatin accessibilityntsuas hauv E14.5 thiab P0 cov qauv, nrog IDRs tseem ceeb hauv ntsuab (qhib) thiab liab (kaw), ua raws li ChiP-seq ntsuas hauv cov cheeb tsam no ntsuas los ntawm Guo et. al. hauv thawj nephron progenitorhlwb(Guo et al., 2021). Lub vaj huam sib luag thib peb qhia txog cov lus piav qhia txog noob caj noob ces (xiav) thiab kwv yees cov cai tswj hwm los ntawm Enhancer Atlas 2.0 (liab), thiab lub vaj huam sib luag thib plaub qhia txog cov kev hloov pauv cov hneev taw taw qhia hauv ATAC-seq cov ntaub ntawv. Lub vaj huam sib luag thib tsib qhia txog nucleosomes (grey) thiab NFRs (liab), thiab lub vaj huam sib luag hauv qab qhia txog kev txuag. A) 1MB cheeb tsam uas muaj cov noob lub cev rau Eya1 thiab muaj peev xwm Eya1 txhim kho kwv yees li 325kb ntws. B) Ib cheeb tsam 4.5kb uas muaj peev xwm Eya1 enhancer. C) 1MB cheeb tsam uas muaj Pax8 gene locus thiab kev kwv yees ua kom zoo dua. D) Ib cheeb tsam 4.5kb uas muaj cov kev kwv yees ua kom zoo dua qub thiab nws qhov sib tshooj DAR.
Topologically txuam domains pab txheeb xyuas qhov muaj peev xwm txhim kho-miRNA kev sib raug zoo
Topologically txuam domains (TADs) yog megabase-scale cheeb tsam ntawm siab dua-order chromatin lub koom haum uas tshwm sim los ntawm lub ntim ntawm chromatin rau hauv lub nucleus nyob rau hauv peb-dimensional qhov chaw (Dixon li al., 2012), thiab enhancer-promoter kev sib cuam tshuam feem ntau txwv rau cov ntsiab lus hauv tib TAD (Symmons et al., 2014). Peb kwv yees TADs los ntawm nephron progenitors siv annotations generated los ntawm nas embryonic qia hlwb (ESCs) (Y. Wang li al., 2018). Txhawm rau tshuaj xyuas cov cheeb tsam cis-kev tswj hwm ntawm miRNA, peb cuam tshuam cov kev hloov pauv hauvchromatin accessibilitynrog sib txawv qhia miRNA hauv tib TAD. Zuag qhia tag nrho, 42,778 cov cheeb tsam tshwj xeeb nkag tau (kwv yees li 92 feem pua) yog nyob rau hauv cov ntawv teev npe TADs, thiab ntawm 3 0,626 (kwv yees 72 feem pua) tsis sib tshooj ib tus neeg txhawb nqa paub. Qhov no suav nrog 2,103 DARs, nrog 1,180 thiab 923 qhib thiab kaw lub sijhawm, feem. Kaum tsib qhib DARs qhia TAD nrog ib lossis ntau dua miRNAs nrog kev nthuav qhia ntxiv ntawm E14.5 thiab P0, thiab cuaj kaw DARs qhia TAD nrog ib lossis ntau dua miRNA nrog kev qhia qis. Daim duab 6 piav qhia txog txoj hauv kev no thiab qhia txog qhov kev ncua deb ntawm DARs thiab lawv qhov sib tw miRNA, uas muaj li ntawm 27 kb mus txog 1 mb (nruab nrab: 447 kb). Table 1 cov ntsiab lus teeb tsa ntawm qhov ua tau DAR - miRNA kev sib raug zoo, nrog ib qho DAR teev rau ib qho sib tw miRNA.
Ntawm cov miRNA-enhancer khub annotated, peb nco txog peb qhov kev txaus siab. Ua ntej, qhov qhib DAR tau txheeb xyuas 120 kb downstream ntawm let-7c-5p thiab 73 kb downstream ntawm miR-125b-5p, uas yog txuam nrog rau Six2 khi qhov chaw nyob rau hauv ib tug yav dhau los luam tawm E16.5 nephron progenitor Chip-seq dataset (Daim duab 7A) (Guo li al., 2021). Cia-7c-5p yog ib qho ntawm cov lus tshaj tawm tshaj tawm cia-7 miRNAs peb tshawb pom, thiab cia{17}} tsev neeg tau paub los tseem cem Lin28btranscripts (Slack & Ruvkun, 1997; Zhou et al., 2011). Peb nco ntsoov tias Hmga2 thiab Meis2 kuj raug txheeb xyuas tias muaj peev xwm tshiab lub hom phiaj ntawm let-7 tsev neeg hauv nephron progenitors (Daim duab 2). miR-125b-5p kuj tau pom tias yuav txwv Lin28 cov ntawv sau tseg hauv nas hematopoietic thiab progenitor cell hom (Chaudhuri et al., 2012). Thib ob, miR-9-3p yog qhov rov qab ntxiv rau miR-9-5p, miRNA uas tsis ntev los no tau qhia los tiv thaivraumfibrosis los ntawm kev tsom mus rau txoj kev metabolic (Fierro-Fernández li al., 2020). Peb kuaj pom qhov ntau dua 10-fold txo qis hauv miR-9-3 cov ntawv teev lus ntawm E14.5 thiab P0 nephron progenitors, thiab peb tau txheeb xyuas qhov muaj peev xwm tswj tau kwv yees li 249kb deb (Daim duab 7B). Peb soj ntsuam cov kev hloov pauv cov hneev taw rau Snai1 thiab Snai2 hauv DAR cuam tshuam, uas tau pom los txhawb kev hloov pauv ntawm epithelial-to-mesenchymal (EMT) (Sundararajan li al., 2019). Thaum kawg, peb tau sib tw miR-181a-2-3p mus rau qhov muaj peev xwm intergenic cis-regulatory locus 131kb sab sauv ntawm chromosome 2, qhov uas peb sau tseg qhov kev hloov pauv qhov taw qhia rau qhov hloov pauv Wt1, tus tswj hwm tseem ceeb ntawm nephron progenitor ciaj sia taus (Kreidberg li al., 1993), thiab Six2 khi nyob rau hauv ib tug luam tawm E16.5 nephron progenitor Chip-seq dataset (Daim duab 7C) ) (Guo li al., 2021).
Table 1. miRNA kev hloov pauv, sib phim rau ib lossis ntau dua DAR hauv tib TAD.
Daim duab 6. Nrhiav tus neeg sib tw ua kom zoo dua-lub hom phiaj-miRNA pairs.A) Flow chart denoting how regions of accessibility are prioritized for possible regulation of miRNA expression. B) Genomic qhov chaw ntawm txhua qhov tshuaj ntsuam miRNA txheeb ze rau lawv cov neeg sib tw ua kom zoo dua qub (txiv kab ntxwv ntsug kab txaij). Cov nplai ntawm lawv cov log2 fold hloov pauv tau pom ntawm y-axis, thiab miRNA nrog nce lossis txo qhov qhia tau hais txog ntsuab thiab liab, feem.
Daim duab 7. Plaub lub peev xwm txhim kho-lub hom phiaj miRNA khub.TADs yog qhov tseem ceeb hauv grey, ua raws cov ntsiab lus pib, thiab kos rau hauv tib qhov ntsuas los qhia txog qhov sib txheeb loj. Paub cov noob los ntawm Ensembl tau pom hauv ntshav, thiab cov cheeb tsam tsaus nti qhia exons. Cov txheeb ze nkag tau ntawm E14.5 thiab P0 yog qhia raws li cov phiaj xwm bar saum toj no thiab hauv qab kab rov tav dub, feem, nrog rau hauv cov phiaj xwm inset qhia txog pawg hauv DAR uas tau teev tseg nrog rau ChiP-seq cov ntaub ntawv luam tawm los ntawm Guo et . al. (Guo et al., 2021). Cov kev hloov pauv cov hneev taw taw qhia los ntawm HINT tau teev nyob rau hauv cov ntawv cim txiv kab ntxwv. Cov phiaj xwm hauv cov vaj huam sib luag sib cais ntawm sab xis qhia txog miRNA kev qhia (ntsuab) thiab cov xwm txheej zoo li Tn5 tso rau hauv cov cheeb tsam uas tau tsim kho (txiv kab ntxwv). A) Tus cia-7 cov neeg hauv tsev neeg cia-7c-5p tau nthuav tawm heev hauvnephron progenitor hlwbthiab tau sib phim rau "peak_54636" 119kb tam sim ntawd hauv thaj av sib txawv. B) miR-9-3p yog phim rau DAR 249kb deb. C) miR-181a 2-3p yog nyob 131kb deb ntawm "peak_5887," ib qho tuaj yeem ua kom muaj kev sib txuas.
Kev sib tham
Hauv txoj kev tshawb no, peb tau tshawb nrhiav ib txhij txheeb xyuas cov tshiab miRNA uas ua rau muaj kev cuam tshuam ntawm nephrogenesis, nrog rau cov peev txheej cis-kev tswj hwm. Peb tau txheeb xyuas 114 miRNAs uas sib txawv hauv nephron progenitor cells, nrog rau 2,103 DARs, hauv E14.5 piv rau P0lub raum. Peb soj ntsuam ntau yam uas kwv yees tus tshiab putative enhancers rau cov noob Eya1 thiab Pax8 nyob rau hauvnephron progenitor hlwb. Thaum kawg, peb txheeb xyuas qhov sib xwschromatin accessibilitythiab miRNA kev hloov pauv rau 33 khub ntawm miRNAs thiab DARs, suav nrog cia-7-5p, miR-125b-5p, miR-181a-2-3p, thiab miR-9-3p, thiab tam sim no lub hom phiaj cov noob tau nthuav tawm hauv nephron progenitors rau qhov sib txawv ntawm miRNAs. Ua ke, peb cov ntaub ntawv qhia tau hais tias kev hloov pauv hauv qhov tseeb rau nephron progenitors thiab nyob ntawm theem ntawm nephrogenesis tau cuam tshuam nyob rau hauv progenitor cell chromatin ib puag ncig thiab hauv nws cov miRNA transcriptome.
Ntawm cov kev hloov pauv uas peb soj ntsuam hauv nephron progenitor miRNA transcriptome, peb tau sau tseg tias muaj kev nthuav dav ntau ntxiv nyob thoob plaws lub let{0}} tsev neeg raws li progenitors hnub nyoog. Qhov no zoo ib yam nrog cov ntaub ntawv dhau los: Lin28b protein thiab let-7 tsev neeg ntawm miRNAs yog kev sib nrig sib antagonistic thiab tsim ib tug bistable hloov uas tswj cia -7 qhia nyob rau hauv cov tsiaj (Zhu li al., 2011), thiab nyob rau hauv nephron progenitors, hloov mus rau kev nce ntxiv cia -7 qhia ncaj qha cuam tshuam rau lub sijhawm ntawm kev txiav tawm ntawm nephrogenesis (Yermalovich li al., 2019). Peb kuj tau soj ntsuam kev nthuav qhia ntau ntxiv ntawm miR-125b-5p, uas tseem paub txog kev hais tawm ntawm Lin28b hauv lwm cov ntsiab lus ntawm tes (Chaudhuri li al., 2012; Potenza et al., 2017). Cov cia-7 tsev neeg thiab miR-125b-5p yog ntawm 114 miRNA uas txawv ntawm E14.5 thiab P0 hauv nephron progenitors, uas qhia txog lub luag haujlwm ntawm miRNAs hauv nephron progenitor txoj kev loj hlob tsis txwv rau cia -7/Lin28b. Piv txwv li, miRNAs uas txo qis hauv kev nthuav qhia suav nrog ob tus tswv cuab ntawm miR-200 tsev neeg, uas tau tshaj tawm los ua lub luag haujlwm hauv podocyte sib txawv (Z. Li li al., 2016): miR-200b -3p thiab miR-429-3p. Ntxiv mus, peb pom qhov txo qis hauv kev qhia ntawm miR-126a 5p, uas paub txog kev txhawb nqa vasculogenesis (Schober et al., 2014; S. Wang et al., 2008). Qhov no tuaj yeem cuam tshuam txog cov kab mob mesenchymal ntawm vasculogenic thiab nephron progenitors, thiab kev cog lus ntawm ib qho ntawm cov hlwb los ua nephron progenitors.
Txoj hauv kev soj ntsuam ntawm kev kwv yees cov hom phiaj ntawm kev hloov miRNAs siv DIANA miRPath (Vlachos li al., 2015) qhia kev txhawb nqa MAPK thiab TGF- signaling, ob qho tib si tseem ceeb ntawm nephron progenitors (Hilliard et al., 2019; Ihermann-Hella 2018, et al. ). Ob peb lwm txoj hauv kev KEGG kuj tseem muaj ntau dhau ntawm cov kev kwv yees cov hom phiaj ntawm kev tswj hwm miRNAs, suav nrog txoj hauv kev cuam tshuam nrog cov kab mob extracellular, actin cytoskeleton, thiab focal adhesion: txhua qhov tseem ceeb ntawm kev tsiv teb tsaws ntawm tes, kev sib txawv, ceg, txuas, polarization, thiab kev loj hlob. (Rozario & Desimone, 2010). ECM cov noob caj noob ces yog tsom los ntawm qee qhov kev tshaj tawm thiab nthuav dav miRNAs uas peb pom, suav nrog cov ntawv tshaj tawm uas peb pom tau: miR-196a-5p. miR-196a-5p paub tias yog hom phiaj Col1a1, Col1a2, thiab Col3a1-cov noob uas tau tswj hwm hauv Pax2-deficient nephron progenitors uas dhau los hloov pauv mus rau hauv lub raum stroma (Naiman thiab ib., 2017). Yog li, nce miR-196a-5kev tshaj tawm tuaj yeem muaj lub luag haujlwm hauv kev txhawb nqa cov kab lis kev cai raws li cov neeg laus muaj hnub nyoog. Tsis tas li ntawd, peb txheeb xyuas qhov muaj peev xwm tshiab ntawm miRNA-mRNA lub hom phiaj kev sib cuam tshuam uas yuav ua rau muaj kev cuam tshuam ntawm nephrogenesis, suav nrog kev tswj hwm ntawm let{25}} tsev neeg uas tuaj yeem tsom Hmga2 thiab Meis2 (txuas nrog kev loj hlob ntawm tes) (Abruzzese li al. ., 2019; Fusco & Fedele, 2007; Lam et al., 2014; Mugford et al., 2009) as nephron progenitors age. Hmga2 twb tsis paub lub luag haujlwm hauvraumkev loj hlob. Txawm li cas los xij, Meis2 tau xav tias muaj kev sib tshooj ua haujlwm nrog qhov cuam tshuam txog kev hloov pauv Meis1, uas paub tias yuav tsum muaj rau kev loj hlob ntawm lub raum (Hisa li al., 2004). Peb kuj soj ntsuam cov kev cai qis ntawm miRs (miR-34b-5p, miR-983p, thiab miR-200a-3p) uas tej zaum yuav lub hom phiaj Jag1, uas paub tias yuav tsum tau tswj hwm thiab tseem ceeb hauv kev sib txawv ntawm nephron (Chung li al., 2016).
Nrog rau kev hloov pauv miRNA transcriptome, peb ib txhij ntsuas kev hloov pauv hauv nephron progenitor cell's chromatin ib puag ncig ntawm E14.5 thiab P0. Peb piav qhia txog ob qhov kev hloov kho tshiab rau Eya1 thiab Pax8, raws li (i) lawv qhov sib thooj rau cov neeg txhawb nqa cov noob no, (ii) lawv cov kev hloov pauv hauvchromatin accessibilitythaum lub sij hawm txoj kev loj hlob nyob rau hauv txoj kab nrog pseudotime gene qhia kev hloov thaum lub sij hawm sib txawv, (iii) hneev taw ntawmraumkev txhim kho cov ntaub ntawv sau tseg, (iv) kev txuag ib ntus, (v) pov thawj ntawm kev ua kom muaj zog hauv lwm cov ntaub so ntswg / cell hom los ntawm Enhancer Atlas2.0 (Gao & Qian, 2020), thiab (vi) Chip-seq cov ntaub ntawv hauv E16 .5 nephron progenitors rau lub cim ntawm kev ua kom muaj zog (H3K27ac) thiab kev sib txuas lus sib txuas ntawm Ctnnb1, Lef1, Six2, thiab Tcf7. Peb pom qhov txo qis ntawm chromatin nkag mus rau Eya1 enhancer, uas yog raws li Eya1 lub luag hauj lwm hauv kev txhawb nqa nephron progenitor multipotency thiab nrog cov lus ceeb toom tias poob ntawm Eya1 tshwm sim nyob rau hauv lub epithelialization ntawm progenitor pej xeem (J. Xu li al., 2014; PX Xu et al., 1999). Pax8 thiab nws homolog Pax2 pab txhawb rau cov kab mob tshwj xeeb ntawm nephron progenitors (Bouchard li al., 2002), thiab nephron progenitors yam tsis muaj tsawg kawg yog ib qho ntawm cov noob no yuav tsis raug MET (Narlis li al., 2007). Qhov nce chromatin nkag tau peb kuaj pom rau cov tshuaj Pax8 txhim kho yog ua raws li Pax8 qhia ntau ntxiv hauv lub raum epithelia raws li nephrons sib txawv.
Enrichment analysis (McLean et al., 2010) ntawm DARs qhia "Txoj Cai ntawm Epithelial Differentiation" raws li lub ntsiab lus tseem ceeb tshaj plaws ntawm cov cheeb tsam qhib chromatin, qhia tias DARs peb tshawb pom tuaj yeem muaj cov yam ntxwv ntawm kev tsav tsheb lossis teb rau cov cim rau kev sib txawv thiab kev loj hlob ntawmnephron progenitor hlwbnyob rau hauv epithelial cell hom. Notch signaling yog ib qho tseem ceeb tshaj plaws ntawm GO cov ntsiab lus uas tau txheeb xyuas ntawm cov qhib DARs, thiab hauv nephron progenitors, txoj hauv kev no paub txog kev txwv rau Six2 qhia thiab txhawb nqa nephron progenitor cell sib txawv (Chung et al., 2016). Ntawm txoj kev no txoj kev cuam tshuam cov noob yog Jag1, ib qho tseem ceeb ligand hauv Notch signaling uas nws cov lus tau tshaj tawm tias muaj kev sib cuam tshuam nrog kev qhia ntawm Six2 thiab Hes1 (Chung et al., 2016). Peb nco ntsoov tias Jag1 yog lub hom phiaj sau tseg rau peb lub miRNAs nrog kev nthuav qhia ntxiv (miR34b-5p, miR98-3p, thiab miR200a-3p, Daim duab 2). Thaum kawg, peb pom kev txhawb nqa rau cov noob uas cuam tshuam nrog kev kho cov hlab ntsha thiab morphogenesis hauv kev qhib DARs, txawm hais tias qhov no zoo li yog qhov tshwm sim ntawm cov noob sib koom ntawm cov kev loj hlob thiab kev sib txawv ntawm cov epithelial, suav nrog Jag1, Eya1, Hes1, Foxc1, thiab Hey2.
Hloov hauvchromatin accessibilityntawmnephron progenitor hlwbtau ntsuas hauv cov kev tshawb fawb yav dhau los (Guo li al., 2021; Hilliard li al., 2019).
Zuag qhia tag nrho, peb cov txiaj ntsig tau zoo ib yam nrog cov kev tshawb fawb yav dhau los, hais txog Notchsignaling thiab Pax8- ntsig txog cov noob. Tseem ceeb, txawm li cas los xij, muaj qee qhov kev sim sib txawv hauv cov kev tshawb fawb ua ntej no thaum piv rau daim ntawv qhia no. Kev rho tawm ntawm GFP-expressing nephron progenitors siv lub fluorescence-activated cell los ntawm Six2-TGC transgene reporter nas muaj feem cuam tshuam los ntawm qhov kev tshawb pom tias cov allele no tau tshaj tawm cov nephron tsawg ntawm kwv yees li 45 feem pua (Hilliard li al. , 2019; Volovelsky et al., 2018). Tsis tas li ntawd, kab lis kev cai nephron progenitors (loj hlob nrog qis lossis siab koob tshuaj CHIR) tau pom tias muaj cov ntaub ntawv sib txawv los ntawm cov thawj nephron progenitors, qhia tias lawv cov chromatin nkag tau kuj yuav txawv (Guo li al., 2021; Hilliard li al., 2019). Txij li thaum lub hom phiaj ntawm peb txoj kev tshawb fawb yog kom nkag siab tias yuav ua li cas hloov pauv hauv chromatin toj roob hauv pes cuam tshuam miRNA kev qhia thaum ntxov piv rau nephron progenitors lig, kev tsom xam ntawm thawj nephron progenitors yog tsim nyog.
Cistanche zoo dua rau lub raum
Los ntawm kev sib txuas cov kev hloov pauv no hauv ib puag ncig chromatin nrog kev hloov pauv hauv miRNA kev qhia, peb cuam tshuam ntau qhov tshiab putative miRNA-enhancer khub hauv kev txiav tawm ntawm nephrogenesis rau kev kawm ntxiv. MiRNAs miR-125a-5p thiab cia-7c-5p, piv txwv li, pom qhov nce ntxiv ntawm E14.5 thiab P0 , thiab qhov no tau rov hais dua los ntawm kev nkag tsis tau ntawm DAR (chr16:77,719,058 77,720,407) hauv tib TAD. Qhov DAR no kuj tseem cuam tshuam nrog rau Six2 qhov chaw sib txuas (Guo li al., 2021), thiab tuaj yeem qhia txog qhov kev txhim kho uas cuam tshuam rau kev qhia ntawm miR-125a-5p thiab/los yog cia{{18} }c-5p, uas nyob rau hauv lem tuaj yeem txhawb kev sib txawv los ntawm kev tsim txom ntawm Lin28b. Ib yam li ntawd, peb pom qhov ua tau zoo rau miR 181a, uas paub txog kev hloov kho renin qhia hauv tib neeg (Marques li al., 2015) thiab tau suav tias yog lub hom phiaj kho mob kom txo tau cov tshuaj nephrotoxicity vim nws lub peev xwm los tawm tsam BIRC6 thiab apoptosis (Liu et ib., 2018). Thaum kawg, peb pom qhov kaw DAR cuam tshuam nrog miR-9-3p uas muaj qhov hloov pauv qhov taw qhia rau Wnt signaling-responsive proteins Snai1 thiab Snai2.miR-9-3p's reverse complement, miR-9-5p, paub tiv thaiv los ntawmraumfibrosis (Fierro-Fernández li al., 2020), Snai1 thiab Snai2 tau pom los txhawb kev mob qog noj ntshav hauv nas cov qog nqaij hlav cancer (Olmeda li al., 2008) thiab txhawb kev hloov pauv epithelial-to-mesenchymal (EMT) hauv cov neeg mob qog noj ntshav. (Sundararajan et al., 2019), thaum miR-9 paub los txhawb kev tsim cov tshuaj angiogenesis thiab kev tsiv teb tsaws ntawm cov qog endothelial hlwb (Zhuang li al., 2012). Ib lub tswv yim yuav muaj nyob rau hauv uas nce Wnt signaling ua rau txo qis ntawm Snai1and Snai2, uas nyob rau hauv lem txo miR-9-3p qhia los txhawb nephron progenitor sib txawv.
Peb txoj kev tshawb fawb tau txheeb xyuas qhov kev hloov pauv hauv thaj chaw chromatin thiab miRNA transcriptome ntawm nephron progenitors thaum lub sij hawm embryonic txoj kev loj hlob, thiab peb tau txheeb xyuas cov neeg sib tw cis-txoj cai cheeb tsam thiab miRNA uas yuav ua lub luag haujlwm hauv kev txiav tawm ntawm nephrogenesis. Enrichment analyses rau ob qho tib si mRNA-seq thiab ATAC-seq cov ntaub ntawv muab cov ntsiab lus ntxim nyiam rau cov txheej txheem uas yuav koom nrog, xws li txoj hauv kev uas tswj hwm nephron progenitor sib txawv, suav nrog Notch thiab TGF-. Peb kuj tau tshaj tawm ob txoj cai tswj hwm tshiab rau Eya1 thiab Pax8, cov noob paub tias tseem ceeb hauvraumtxoj kev loj hlob, thiab muaj peev xwm tswj kev ua ntu zus rau cov miRNA tseem ceeb uas cuam tshuam rau kev txiav tawm ntawm nephrogenesis.
Cov txheej txheem
Nas hom
Wildtype CD-1 cov poj niam cev xeeb tub tau txais los ntawm Charles River Laboratories, Inc. (Wilmington, MA, USA, RRID: MGI: 5659424) thiablub raumtau sau los ntawm litters ntawm embryonic hnub E14.5 los yog P0. Txhua tus tsiaj tau nyob hauv vivarium ntawm Rangos Research Center ntawm UPMC Children's Hospital of Pittsburgh (Pittsburgh, PA, USA) thiab tag nrho cov kev sim tsiaj tau ua raws li cov cai ntawm Pawg Saib Xyuas Tsiaj Tsiaj thiab Siv Pawg ntawm University of Pittsburgh. Kev sib deev rau txhua lub embryo lossis pup raug txheeb xyuas los ntawm kev ua PCR ntawm genomic DNA cais los ntawm tus Tsov tus tw clippings siv cov hauv qab no primers rau Y-chromosome gene Sry: SryF 5ʹ-GATGATTTGAGTGGAAATGTGAAGGTA-3ʹ thiab SryR 5ʹ-CTATATGTTAs{6} yav dhau los luam tawm (McFarlane li al., 2013).
Me me RNA sequencing thiab tsom xam
Tag nrho RNA cais los ntawmnephron progenitor hlwbseem tom qab tshem tawm ATAC-seq ntawm tes ntu tau xa mus rau Health Sciences Sequencing Core ntawm UPMC Children's Hospital of Pittsburgh rau kev npaj tsev qiv ntawv siv QIAseq miRNA lub tsev qiv ntawv npaj cov khoom siv (Qiagen 3315{{10}}}2). Tib-kawg sequencing ntawm mRNA-seq cov tsev qiv ntawv tau ua nyob rau ntawm Illumina NextSeq500, thiab cov tsev qiv ntawv tau ua raws li qhov tob ntawm kwv yees li 50 lab nyeem ib zaug hauv ib lub tsev qiv ntawv. Cov ntawv nyeem tau txuas mus rau tus nas mm10 genome siv pob Rsubread (Liao li al., 2019) (version 2.0.1), thiab sau tseg rau cov paub miRNA teev nyob rau hauv miRBase (version 22) (Kozomara & Griffiths-Jones, 2014) nrog Rsubread pob lub featureCounts muaj nuj nqi. Kev qhia txawv ntawm miRNA paub tau raug ntsuas siv DESeq2 R pob (version 1.26.0) (Hlub li al., 2014). Ib feem ntawm cov ntawv nyeem tau sau tseg rau cov miRNA paub nrog rau feem ntawm cov poj niam embryos ib qho piv txwv tau suav nrog cov cofactors hauv cov qauv kuaj xa mus rau DESeq2. Qhov txawv ntawm miRNA raug txheeb xyuas thaum tswj qhov kev tshawb pom tsis tseeb ntawm 5 feem pua , thiab lawv cov kev taw qhia ntawm kev hloov pauv (nce thiab txo qis) tau txiav txim siab raws li lawv qhov kev hloov pauv tus nqi ("nce" qhia ntau dua ntawm P0 piv nrog E14.5) . Txhim kho txoj hauv kev tswj hwm ntawm kev kwv yees cov hom phiaj ntawm miRNA nrog kev nthuav dav thiab txo qis tau suav nrog siv DIANA cov cuab yeej miRPath version 3.0 (Vlachos li al., 2015). Cov ntaub ntawv kwv yees lub hom phiaj rau miRNA tau muab los ntawm TargetScan (Agarwal li al., 2015) thiab miRDB (Y. Chen & Wang, 2020) repositories.
Nephron progenitor cais tawm
Lub raumtau muab tshem tawm los ntawm litters ntawm {{0}} E14.5 embryos lossis P0 menyuam dev, thiab txhua lub litter raug suav tias yog ib qho qauv. Ib lub raum rau ib tus qauv tau siv rau tag nrho RNA kev rho tawm los siv ua "tag nrho lub raum tswj" hauv kev tshuaj ntsuam tom ntej (saib hauv qab). Tag nrho ntawm peb cov qauv raug sau rau ntawm txhua lub sijhawm. Cortical hlwb raug cais tawm ntawm txhua tus qauv siv sib nqus-activated cell sorting (MACS) raws li tau tshaj tawm yav dhau los (Brown li al., 2011). Luv luv, ob lub raum tau txais ib feem ntawm kev zom nrog 2mM collagenase A (Roche 11088793001) thiab 3.5mM pancreatin (Sigma P1625) hauv phosphate-buffered saline (PBS) rau 15 min ntawm 37 degree. Cov tshuaj tiv thaiv kev zom zaub mov tau raug tso tseg siv cov tshuaj txias 100 feem pua fetal bovine serum (FBS), thiab cov kev ncua ntawm tes tau sau thiab rov ua dua nyob rau hauv cov khaub thuas Dulbecco's phosphate-buffered saline (DPBS) nrog 1mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor (Sigma 10-10-10). Cov hlwb raug ntxuav thiab rov ua dua nyob rau hauv qhov dej khov-txias cais tsis muaj 2 feem pua FBS hauv PBS, tom qab ntawd incubated nrog cov hlaws sib nqus (Dynabeads FlowComp Flexi cov khoom, ThermoFisher 11061D) biotinylated rau cov tshuaj tiv thaiv rau Integrin alpha, 8 R4 & 7. siv DSB-X Biotin Protein Labeling Kit (ThermoFisher D-20655). Nephron progenitors khi rau cov hlaws dai no tau raug cais raws li tau tshaj tawm yav dhau los (Hemker li al., 2020) thiab sib sau ua ke hauv ob lub raum rau txhua tus qauv. Ib qho aliquot ntawm 50,000 nephron progenitor cells tau ua tiav tam sim los ntawmchromatin accessibilitytsev qiv ntawv npaj raws tu qauv (saib hauv qab), thiab tag nrho RNA tau muab rho tawm los ntawm qhov seem ntawm tes raug ncua siv Qiagen miRNeasy Mini Kit (Qiagen 217004).
Kom paub meej tias enrichment ntawmnephron progenitor hlwbtxheeb ze rau lwm hom cell, tag nrho RNA raug cais tawm ntawm nephron progenitors thiab los ntawm tag nrholub raum(tag nrho lub raum tswj, saib saum toj), thiab ntau qhov rov qab-transcription PCR (qRT-PCR) rau cov cim hauv qab no tau ua: nephron progenitor markers Six2 thiab Cited1 (L. Huang li al., 2016), thiab rau Lhx1, Pdgfr , Pecan, thiab Caleb uas cim lub raum vesicle (Brunskill li al., 2014), raum stroma (S. Chen li al., 2015), endothelial (Kobayashi li al., 2008), thiab ureteric bud (Cebrian li al., 2014a ) cell, feem; kuj pom daim duab ntxiv 1. Gapdh (Barber et al., 2005) tau siv los ua ib lub tsev tu noob thiab cov txheeb ze txheeb ze raug xam los ntawm 2-ΔΔCt txoj kev (Livak & Schmittgen, 2001). Primers rau cov ntawv sau tseg no suav nrog hauv Table 2.
Table 2. RT-PCR primers.
Chromatin accessibilitykev npaj tsev qiv ntawv
Kwv yees li 50,000nephron progenitor hlwbtau siv los tsim txhua lub tsev qiv ntawv sib txuas rau ATAC-seq siv Nextera DNA Flex Library Prep Kit (Illumina FC-121-1030) nrog kev hloov kho raws li ib txoj kev luam tawm yav dhau los (Buenrostro li al., 2013). Hauv luv luv, cov hlwb raug tshem tawm hauv cov dej khov-txias uas tsis yog-ionic lysis tsis muaj 10mM Tris, 10mM NaCl, 3mM MgCl2, 1mM PMSF protease inhibitor, thiab 0.05 feem pua Triton X-100 los ntawm ntim rau 10 feeb rau lyse cell membranes thaum tawm hauv nuclear membranes tsis zoo. Intact nephron progenitor nuclei raug tshem tawm hauv kev hloov pauv tsis muaj cov enzyme transposase thiab incubated ntawm 37 degree rau 30 feeb. Dawb genomic DNA tso tawm los ntawm cov txheej txheem kev hloov pauv tau ua kom huv si siv MinElute PCR Purification cov khoom siv (Qiagen 28004) thiab tom qab ntawd muab ntsuas siv rau pem hauv ntej (i7) thiab thim rov qab (i5) index primers los ntawm Nextera Index Kit (Illumina FC-121-1011). Index ligation thiab fragment amplification tau ua tiav siv txoj kev PCR amplification thermal cycling program.
Txhawm rau txiav txim siab qhov zoo tshaj plaws ntawm lub voj voog uas xav tau rau lub tsev qiv ntawv ATAC-seq, qPCR sab cov tshuaj tiv thaiv tau ua tiav siv SsoAdvanced SYBR Supermix (BioRad 1725274) thiab 96-zoo C100 Thermal Cycler (Bio-Rad) los xam cov normalized tus nqi tshaj tawm (Rn) rau txhua lub voj voog. Lub voj voog tus naj npawb ntawm cov tshuaj tiv thaiv mus txog ib feem peb ntawm nws qhov siab tshaj plaws fluorescence tau pom tias yog qhov zoo tshaj plaws ntawm cov voj voog ntawm kev nthuav dav ntxiv, thiab qhov seem seem ntawm qhov ntsuas ATAC-seq hloov pauv cov tshuaj tiv thaiv tau raug rau tus lej ntawm cov voj voog ntxiv hauv qhov kawg amplification. qhov kev pab cuam. Cov tsev qiv ntawv kawg tau ua kom huv si siv Ampure XP magnetic hlaws (Beckman Coulter A63881).
Chromatin accessibilityua ke
Paired-end sequencing ntawm lub tsev qiv ntawv ATAC-seq tau ua tiav ntawm Illumina NextSeq500 los ntawm Health Sciences Sequencing Core ntawm UPMC Children's Hospital of Pittsburgh, multiplexed nrog cov tsev qiv ntawv xav tau kom tau txais 90 lab ua khub-kawg nyeem ib tus qauv . Cov ntawv nyeem tau raug txiav zoo siv TrimGalore (BabrahamLab, 2014) (version 0.4.3) hauv "--paired" hom thiab nrog rau lwm yam kev teeb tsa. Cov ntawv nyeem tau ua raws li mm10 genome (Church li al., 2011) siv Bowtie2 (Langmead li al., 2009) (version 2.3.1) nrog rau cov chaw "--local -q -X 2000 -- m". Cov ntawv nyeem uas tshwm sim los ntawm PCR duplicates ntawm tib DNA tawg tau raug cim siv Picard Cov Cuab Yeej '(Broad Institute, 2016) MarkDuplicates muaj nuj nqi (version 2.10.9) nrog rau qhov chaw pib. Nyeem ntawv uas tsis meej pem rau ntau tshaj ib qho chaw hauv genome tau muab tso rau hauv ib qho ntawm cov chaw no yog tias muaj tsawg dua plaub qhov muaj peev xwm, thiab raug tshem tawm (ENCODE, nd) (siv cov ntawv Python muaj nyob ntawm https://github. com/kundajelab/atac_dnase_pipelines, (Lee, 2017) nrog rau cov chaw "--paired-end -k"). Samtools (H. Li et al., 2009) (version 1.3.1) yog siv los lim tawm cov ntaub ntawv duplicated, unmapped, orphaned, thiab mitochondrial nyeem, nrog rau kev sorting thiab indexing ntawm BAM cov ntaub ntawv. Kev tswj xyuas zoo ntawm ATAC-seq cov ntaub ntawv tau ua raws li ENCODE qhov project's ATAC-seq cov lus qhia (ENCODE, nd), thiab cov tsev qiv ntawv coj mus kuaj tau random down-sampled rau txhua tus muaj 31 lab ua khub-kawg nyeem kom zoo rau qhov sib txawv ntawm cov tsev qiv ntawv loj.
Cistanche tuaj yeem tiv thaiv kab mob raum
Txheeb xyuas cov cheeb tsam chromatin nkag tau
BAM cov ntaub ntawv uas muaj cov ntawv nyeem ATAC-seq tau hloov pauv mus ua khub-kawg BED hom siv Bedtools' (Quinlan & Hall, 201{{20}}) (version 2.26.0) 'bombed ' ua haujlwm nrog cov chaw "- txaj pe -mate1". Cov cheeb tsam ntawm cov chromatin siv tau raug txheeb xyuas siv tus qauv-raws li Kev Ntsuas ntawm ChiP-seq cuab tam (MACS2, version 2.1.1.20160309) broad peak calling algorithm (Zhang et al., 2008), nrog rau cov chaw "--format BEDPE {{ 18}}g mm -p 0. Txhawm rau txheeb xyuas cov cheeb tsam uas muaj kev ntseeg siab siab, kev sib piv ua khub (ntawm txhua qhov kev sib txuas ntawm cov ntawv luam tawm los ntawm lub sijhawm muab) tau ua thiab qhov kev tshawb pom tsis pom tseeb (IDR) (Q. Li et al., 2011) tau suav nrog kev siv pej xeem. Python kev siv (GitHub https://github.com/nboley/idr) (Boleu et al., 2015) nrog cov chaw "--input-file-type broad peak --rank p.value {{ 35}}soft-IDR-threshold 0.1 --cov ntaub ntawv-hom txaj". Tshwj xeeb, rau txhua lub sij hawm taw tes ib cov ntaub ntawv BED concatenated ntawm tag nrho cov cheeb tsam nkag tau raug xa los ntawm "--peak-list" kev sib cav rau txhua qhov kev sib piv (piv txwv li, thaum muab ob qho piv txwv E14.5, tag nrho E14.5 broad peaks nruab nrab ntawm tag nrho peb replicates yog siv). Cov cheeb tsam ntawm kev nkag mus tau zoo ib yam los ntawm IDR nyob rau hauv tsawg kawg yog ob qho kev sib txuas ua ke sib piv (nrog tsawg kawg ntawm 20 feem pua ntawm kev sib tshooj) tau muab tso ua ke rau hauv ib qho kev sib koom ua ke ntawm thaj chaw muaj kev ntseeg siab ("IDR regions").
Kev hloov pauv hauv thaj chaw chromatin nkag tau
Kev nkag mus tau ntawm txhua cheeb tsam IDR nyob rau hauv ib qho piv txwv tau ntsuas los ntawm kev suav cov xwm txheej hloov pauv uas tshwm sim thaum normalizing rau qib GC-cov ntsiab lus hla cov qauv. Qhov no tau ua tiav los ntawm kev siv tsab ntawv kev cai (muaj nyob rau ntawm qhov chaw khaws software nrog, saib cov ntaub ntawv ntxiv). Cov kev hloov pauv hauv qhov kev siv tau no tau txiav txim siab los ntawm kev sib piv cov txiaj ntsig ntawm lub sijhawm cov ntsiab lus siv Limma-voom (version 3.42.2) software pob (Ritchie li al., 2006). Txhawm rau suav nrog kev sib deev embryo, feem ntawm cov poj niam embryos hauv txhua tus qauv tau suav nrog hauv cofactor hauv cov qauv siv rau kev txheeb xyuas qhov sib txawv, thiab nws raug tshem tawm raws li cov nyhuv batch hauv kev pom kev siv lub Limma pob tshem tawm cov nyhuv batch muaj nuj nqi. Cov cheeb tsam nkag tau sib txawv ntawm chromatin (DARs) tau suav tias yog qhov qhib loj heev (nce kev nkag tau ntawm E14.5 thiab P0) lossis kaw (tsawg nkag tau ntawm E14.5 thiab P0) thaum tswj qhov kev tshawb pom tsis tseeb ntawm 10 feem pua. . Functional enrichment analyses of qhib thiab kaw cov cheeb tsam genomic tau txiav txim los ntawm kev xa cov kev tswj hwm mus rau Genomic Regions Enrichment of Annotations Tool (Zoo, muaj nyob ntawm http://great.stanford.edu/public/html/) (McLean li al., 2010 ). Cov lus piav qhia ntawm cov paub thiab kwv yees li kev txhim kho tau muab los ntawm FANTOM5 (Andersson li al., 2014), VISTA (Visel li al., 2007), thiab EnhancerAtlas 2.0 (Gao & Qian, 2020) repositories.
Transcription factor footprints
Kev hloov pauv cov hneev taw tau txheeb xyuas los ntawm kev sib sau tag nrho BAM cov ntaub ntawv los ntawm tib lub sijhawm taw tes, tom qab ntawd khiav Regulatory Genomics Toolbox's Hmm-based Identification of TF Footprints (HINT) software (version 0.12.3), tshwj xeeb tshaj yog tus qauv footprinting rau ATAC -seq cov ntaub ntawv (muaj nyob rau ntawm www.regulatory-genomics.org ) (Z. Li et al., 2018). Cov software no tau khiav nrog kev teeb tsa "right-hint footprinting --organism=mm10 --at-seq --paired-end". Cov hneev taw tau sau tseg nrog cov nas paub txog kev sib khi motifs los ntawm HOCOMOCO 11 database (Kulakovskiy li al., 2018) siv HINT's motif matching function nrog rau cov chaw "txoj cai-motif tsom xam -- kab mob =mm10". Cov hneev taw nrog HINT cov qhab nia qis dua 10 tsis suav nrog. Thaum kawg, cov qauv sib txawv ntawm cov motif-matched hneev taw tau ntsuas siv RGT-HINT qhov kev ua haujlwm sib txawv, nrog rau kev teeb tsa "txoj cai-hint txawv -- kab mob=mm10 --bc {{27} }NC 12 --output-profiles". Cov kev hloov pauv tseem ceeb tau raug txheeb xyuas raws li p-tus nqi txiav tawm ntawm 0.05 (Friedman-Nemeny method).
Cistanche tuaj yeem txhim kho lub raum ua haujlwm
Nucleosomal configuration
Aligned nyeem los ntawm tib lub sij hawm taw tes tau muab sib sau ua ke rau hauv ib qho BAM cov ntaub ntawv, thiab cov pob NucleoATAC (version 0.3.4) tau siv los txheeb xyuas cov qauv hauv kev nyeem ntawv ntev qhia txog ob qho tib si khi nucleosomal dyads thiab ntawm thaj chaw nucleosome-dawb ( NFRs) (Schep et al., 2015). Cov software no tau ua tiav los ntawm kev teeb tsa lub neej ntawd. Nucleosomes tau sau tseg raws li thaj tsam 146bp nyob ib ncig ntawm txhua tus hu ua nucleosome dyad.
Ib leeg-cell RNA qhia
Cov ntaub ntawv qhia ib leeg ntawm tes tau ua tiav rau cov ntawv sau dhau los (BioRxiv https://www.biorxiv.org/content/10.1101/2020.09.16.300293v1) (Bais et al., 2020), thiab tau muab los ntawm Gene Expression Omnibus (GEOID GSE158166). Cov ntaub ntawv ua tiav tau muab tso rau hauv daim duab ntxiv 7 suav nrog hom cell, qib qhia cov noob, qis-dimensional (tSNE) embeddings, thiab pseudotime annotations txheeb ze rau txoj hauv kev tsim los ntawm pawg ntawm proliferating thiab sib txawv nephron progenitors. Cov ntaub ntawv tau nkag mus thiab teeb tsa siv SingleCellExperiment R pob (Lun & Risso, 2019).
miRNA-mRNA kev sib cuam tshuam hauv nephron progenitor cell aging thiab sib txawv
Genes nrog hloov kev qhia ntawm tus kheej-renew, primed, thiab sib txawvnephron progenitor hlwbtau txais los ntawm Supplemental tables 3 thiab 4 luam tawm los ntawm Bais li al.. (FDR< 0.1="" for="" all="" genes,="" 99="" genes="" total).="" mirtarbase="" v8.0="" for="" the="" mouse="" was="" downloaded="" (https://mirtarbase.cuhk.edu.cn/~mirtarbase/mirtarbase_2022/cache/download/8.0/mmu_mt="" i.xls)="" while="" mirdb="" version="" 6.0="" predictions="" were="" obtained="" from="" the="" mid="" b="" website="" (y.="" chen="" &="" wang,="" 2020);="" high-quality="" mirna-to-target-gene="" predicted="" interactions="" with="" a="" minimum="" score="" of="" 90="" were="" considered.="" annotated="" mir="" target="" genes="" and="" differentially="" expressed="" genes="" were="" then="" intersected="" using="" the="" r="" programming="" environment="" to="" generate="" figure="" 2="" and="" supplemental="" table="">
Chromatin immunoprecipitation sequencing nyob rau hauv thawj nephron progenitors
Chromatin immunoprecipitation sequencing (ChIP-seq) cov ntaub ntawv rau -catenin, Lef1, Six2, thiab Tcf7 binding nyob rau hauv thawj nephron progenitors tau muab los ntawm BedGraph cov ntaub ntawv nyob rau hauv lub GEO repository (GSE131119) rau Guo li al., olds 2021 enrichment signal. nyob rau hauv replicate cov qauv.
Kev tshuaj ntsuam xyuas cov cai tswj kev cuam tshuam rau miRNA kev qhia
Txhawm rau tshuaj xyuas qhov muaj peev xwm txhim kho ntawm miRNA, peb tau sau tseg miRNA thiab DARs nrog cov topologically txuam domains (TADs) uas lawv nyob raws mm10 annotations los ntawm nas embryonic qia hlwb, rub tawm los ntawm 3D Genome Browser (muaj nyob ntawm http://3dgenome .fsm.northwestern.edu/) (Y. Wang et al., 2018). Thaj tsam ntawmchromatin accessibilitythiab miRNA raug suav hais tias yog tus neeg sib tw ua kom zoo dua-miRNA khub yog tias lawv nyob hauv tib TAD thiab pom tau tias muaj kev hloov pauv hauv kev nkag mus tau zoo thiab kev nthuav qhia (nce kev nkag mus tau nrog nce miRNA qhia thiab rov ua dua). DARs tsuas yog suav tias yog qhov ua tau "kev tswj hwm cov yam ntxwv" yog tias lawv tsis sib tshooj ib tus paub txhawb lossis exon kawg. Muaj peev xwm ua khub ntawm miRNA thiab cov txheej txheem kev tswj hwm tau ua ntej yog tias lawv 1) poob rau tib lub TAD thiab 2) tau ntsib cov kev hloov pauv tseem ceeb ntawm E14.5 thiab P0 (nce miRNA qhia thiab nce IDR cheeb tsam kev nkag tau, thiab rov ua dua) .
Kev lees paub
Shelby Hemker thiab Abha Bais tau pab txhawb rau kev sim tsim. Sequencing rau ATAC-seq thiab mRNA-seq tau ua los ntawm Health Sciences Sequencing Core ntawm UPMC Children's Hospital of Pittsburgh.
Kev nyiam sib tw
Tsis muaj kev sib tw txaus siab tshaj tawm
Nyiaj txiag
AC tau txais kev txhawb nqa los ntawm cov nyiaj pab los ntawm National Institute of Diabetes thiab Digestive thiabLub raumCov kab mob hauv National Institute of Health (T32DK061296-17). DK tau txais kev txhawb nqa los ntawm National Institute of General Medical Sciences hauv National Institute of Health (R01GM115836). JH tau txais kev txhawb nqa los ntawm cov nyiaj pab los ntawm National Institute of Diabetes thiab Digestive and raum Diseases nyob rau hauv National Institute of Health (R01DK103776 thiab 125015). DMC tau txais kev txhawb nqa los ntawm Nephrotic Syndrome Study Network (NEPTUNE) Career Development Award thiab Children's Hospital of Pittsburgh Research Advisory Council Postdoctoral Fellowship. YP tau txais kev txhawb nqa los ntawm UPMC Children's Hospital of Pittsburgh thiab North American Mitochondrial Disease Consortium.
Cov ntaub ntawv muaj
Predicted nas enhancers tau muab los ntawm FANTOM5 (DOI: 10.1038/nature12787; cov ntaub ntawv muaj nyob ntawm no), Vista (DOI: 10.1093/var/gkl822; cov ntaub ntawv muaj nyob ntawm no), thiab EnhancerAtlas (DOI: 10.1093/var/gkz980; nas cov ntaub ntawv muaj nyob ntawm no); databases. Tus nas mm10 genome thiab nws cov lus piav qhia tau rub tawm ntawm Illumina genomes project (muaj nyob ntawm no). Cov ntaub ntawv tshaj tawm rau kev nqa mm9 kev tswj xyuas rau mm10 tau muab los ntawm UCSC genome browser (DOI: 10.1101/gr.229102; cov ntaub ntawv muaj nyob ntawm no). TAD cov lus piav qhia los ntawm nas embryonic qia hlwb tau muab los ntawm 3D Genome Browser (DOI: 10.1186/s13059-018-1519-9; cov ntaub ntawv muaj nyob ntawm no). Kev kwv yees miRNA cov ntawv sau tseg tau muab los ntawm TargetScan (DOI: 10.7554/eLife.05005, cov ntaub ntawv muaj nyob ntawm no), thiab los ntawm miRDB version 6 (DOI: 10.1093/var/gkz757, cov ntaub ntawv muaj nyob ntawm no). ATAC-seq cov ntaub ntawv suav nrog IDR / DAR qhov chaw, cov lus piav qhia, thiab cov txiaj ntsig sib txawv muaj nyob ntawm GEO (GSE168339), xws li mRNA-seq cov ntaub ntawv suav nrog cov lus piav qhia thiab cov txiaj ntsig sib txawv (GSE168342). Khoos phis tawj code siv los ua cov ntaub ntawv thiab tshuaj xyuas, thiab tsim cov duab muaj nyob ntawm https://bitbucket.org/clugstonA/mirna_enhancers
Cov ntawv sau cia
Abruzzese, MP, Bilotta, MT, Fionda, C., Zingoni, A., Soriani, A., Petrucci, MT, Ricciardi, MR, Molfetta, R., Paolini, R., Santoni, A., & Cippitelli, M . (2019). Lub homeobox transcription factor MEIS2 yog tus tswj hwm ntawm cov qog nqaij hlav qog noj ntshav thiab IMiDs kev ua haujlwm hauv Ntau Myeloma: kev hloov kho los ntawm Bromodomain thiab Extra-Terminal (BET) protein inhibitors. Cell Tuag thiab Kab Mob, 10(4). https://doi.org/10.1038/s41419-019-1562-9
Agarwal, V., Tswb, GW, Nam, JW, & Bartel, DP (2015). Predicting zoo microRNA lub hom phiaj qhov chaw hauv mammalian mRNAs. ELife. https://doi.org/10.7554/eLife.05005
Andersson, R., Gebhard, C., Miguel-Escalada, I., Hoof, I., Bornholdt, J., Boyd, M., Chen, Y., Zhao, X., Schmidl, C., Suzuki, T. ., Ntini, E., Arner, E., Valen, E., Li, K., Schwarzfischer, L., Glatz, D., Raithel, J., Lilje, B., Rapin, N., … Sandelin, A. (2014). Ib qho atlas ntawm active enhancers thoob plaws tib neeg hom cell thiab cov ntaub so ntswg. Xwm, 507(7493), 455–461. https://doi.org/10.1038/nature12787
BabrahamLab. (2014). Trim Galore. Hauv Bioinformatics. http://www.bioinformatics.babraham.ac.uk/projects/trim_galore
Bais, AS, Cerqueira, DM, Clugston, AS, Ho, J., & Kostka, D. (2020). Ib leeg-cell RNA sequencing qhia tawm qhov sib txawv ntawm lub voj voog ua haujlwm hauv cov cell tseem ceeb thaum lub sijhawm nephrogenesis. BioRxiv. https://doi.org/https://doi.org/10.1101/2020.09.16.300293
Barber, RD, Harmer, DW, Coleman, RA, & Clark, BJ (2005). GAPDH raws li lub tsev tu noob: Kev tshuaj xyuas ntawm GAPDH mRNA qhia hauv ib lub vaj huam sib luag ntawm 72 tib neeg cov ntaub so ntswg. Physiological Genomics. https://doi.org/10.1152/physiolgenomics.00025.2005
Bartel, DP (2009). MicroRNA Target Recognition thiab Regulatory Functions. Cell, 136(2), 215–233. https://doi.org/10.1016/j.cell.2009.01.002.MicroRNA
Bertram, JF, Douglas-Denton, RN, Diouf, B., Hughson, MD, & Hoy, WE (2011). Tib neeg nephron tooj: Qhov cuam tshuam rau kev noj qab haus huv thiab kab mob. Pediatric Nephrology, 26(9), 1529–1533. https://doi.org/10.1007/s00467-011-1843-8
Boleu, N., Kundaje, A., Bickel, PJ, & Li, Q. (20}15). Irreproducible Discovery Rate (2.0.3). https://github.com/nboley/idr
Bouchard, M., Souabni, A., Mandler, M., Neubüser, A., & Busslinger, M. (2002). Nephric kab lus tshwj xeeb los ntawm Pax2 thiab Pax8. Cov noob caj noob ces thiab kev loj hlob. https://doi.org/10.1101/gad.240102
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Los ntawm: Phau ntawv Journal Pre-proof
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