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1.
Genes Dev ; 36(1-2): 38-52, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34969824

RESUMO

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

2.
Nat Commun ; 12(1): 6241, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34716321

RESUMO

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation.


Assuntos
Cromatina/química , Leucemia Eritroblástica Aguda/patologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/fisiologia , Células Eritroides/patologia , Leucemia Eritroblástica Aguda/metabolismo , Camundongos Knockout , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Ligação a RNA/genética
3.
Genome Biol ; 22(1): 269, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526084

RESUMO

BACKGROUND: Super-enhancers are clusters of enhancer elements that play critical roles in the maintenance of cell identity. Current investigations on super-enhancers are centered on the established ones in static cell types. How super-enhancers are established during cell differentiation remains obscure. RESULTS: Here, by developing an unbiased approach to systematically analyze the evolving landscape of super-enhancers during cell differentiation in multiple lineages, we discover a general trend where super-enhancers emerge through three distinct temporal patterns: conserved, temporally hierarchical, and de novo. The three types of super-enhancers differ further in association patterns in target gene expression, functional enrichment, and 3D chromatin organization, suggesting they may represent distinct structural and functional subtypes. Furthermore, we dissect the enhancer repertoire within temporally hierarchical super-enhancers, and find enhancers that emerge at early and late stages are enriched with distinct transcription factors, suggesting that the temporal order of establishment of elements within super-enhancers may be directed by underlying DNA sequence. CRISPR-mediated deletion of individual enhancers in differentiated cells shows that both the early- and late-emerged enhancers are indispensable for target gene expression, while in undifferentiated cells early enhancers are involved in the regulation of target genes. CONCLUSIONS: In summary, our analysis highlights the heterogeneity of the super-enhancer population and provides new insights to enhancer functions within super-enhancers.

4.
Cell Stem Cell ; 28(11): 1922-1935.e5, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34529935

RESUMO

Little is known about how interactions of diet, intestinal stem cells (ISCs), and immune cells affect early-stage intestinal tumorigenesis. We show that a high-fat diet (HFD) reduces the expression of the major histocompatibility complex class II (MHC class II) genes in intestinal epithelial cells, including ISCs. This decline in epithelial MHC class II expression in a HFD correlates with reduced intestinal microbiome diversity. Microbial community transfer experiments suggest that epithelial MHC class II expression is regulated by intestinal flora. Mechanistically, pattern recognition receptor (PRR) and interferon-gamma (IFNγ) signaling regulates epithelial MHC class II expression. MHC class II-negative (MHC-II-) ISCs exhibit greater tumor-initiating capacity than their MHC class II-positive (MHC-II+) counterparts upon loss of the tumor suppressor Apc coupled with a HFD, suggesting a role for epithelial MHC class II-mediated immune surveillance in suppressing tumorigenesis. ISC-specific genetic ablation of MHC class II increases tumor burden cell autonomously. Thus, HFD perturbs a microbiome-stem cell-immune cell interaction that contributes to tumor initiation in the intestine.


Assuntos
Antígenos de Histocompatibilidade Classe II , Intestinos , Carcinogênese , Dieta Hiperlipídica , Células Epiteliais , Humanos
5.
Nat Commun ; 12(1): 4991, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404810

RESUMO

Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the ß-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.


Assuntos
Edição de Genes , Hemoglobinas/genética , Hemoglobinas/metabolismo , Sistemas CRISPR-Cas , Cromatina , Cromossomos , Proteínas de Ligação a DNA/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Expressão Gênica , Globinas , Humanos , Mutação , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Globinas beta/genética
6.
Nat Commun ; 12(1): 4439, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290235

RESUMO

The α- and ß-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Ativação Transcricional , Globinas zeta/genética , Acetilação , Animais , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Células Eritroides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , alfa-Globinas/genética
7.
Nat Commun ; 12(1): 3933, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168132

RESUMO

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.


Assuntos
Epigênese Genética , Células Epiteliais/citologia , Complexo Repressor Polycomb 2/genética , Timo/citologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Epiteliais/fisiologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Linfócitos T/fisiologia , Timócitos/citologia , Timócitos/fisiologia , Timo/fisiologia
8.
Med (N Y) ; 2(2): 122-136, 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33688634

RESUMO

Studies of the major hemoglobin disorders, ß-thalassemia and sickle cell disease (SCD), have laid a foundation for molecular medicine. While enormous progress has been made in understanding gene structure and regulation, translating molecular insights to therapy for the many individuals affected with these disorders has been challenging. Advances in three activities have recently converged to bring novel genetic and potentially curative treatments to clinical trials. First, improved lentiviral vectors for gene transfer into hematopoietic stem cells have revived somatic gene therapy for blood disorders. Second, elucidation of regulatory factors and mechanisms that control the normal developmental switch from fetal to adult hemoglobin has provided a route to reactivation of the fetal form for therapy. Third, revolutionary methods of gene engineering permit molecular insights to be leveraged for patients. Here I review how the promise of molecular medicine to bring transformative treatments to the clinical arena is finally being realized.

9.
Nat Genet ; 53(4): 511-520, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649594

RESUMO

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2ß2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb ß-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.


Assuntos
Fator de Ligação a CCAAT/química , Hemoglobina Fetal/genética , Hemoglobina A/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/química , gama-Globinas/química , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Eritropoese/genética , Hemoglobina Fetal/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica , Células HEK293 , Hemoglobina A/metabolismo , Humanos , Modelos Moleculares , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células-Tronco , Globinas beta/química , Globinas beta/genética , Globinas beta/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismo
11.
J Biol Chem ; 295(47): 15797-15809, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-32994224

RESUMO

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb cis-region (cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.


Assuntos
Loci Gênicos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Elementos Reguladores de Transcrição , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Fator 3 de Transcrição de Octâmero/genética
12.
Proc Natl Acad Sci U S A ; 117(35): 21450-21458, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817427

RESUMO

How overall principles of cell-type-specific gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type-specific regulation employs different cis elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer-promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type-specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.


Assuntos
Fatores Etários , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Cromatina , Elementos Facilitadores Genéticos/genética , Eritroblastos , Eritropoese/fisiologia , Feminino , Expressão Gênica , Genômica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética
14.
Cell ; 181(6): 1410-1422.e27, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32413320

RESUMO

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.


Assuntos
Sistemas CRISPR-Cas/genética , Linhagem da Célula/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Transcriptoma/genética , Animais , Linhagem Celular , Feminino , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/fisiologia , Masculino , Camundongos , Transdução Genética/métodos
15.
Genome Biol ; 21(1): 59, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32138752

RESUMO

The spatiotemporal control of 3D genome is fundamental for gene regulation, yet it remains challenging to profile high-resolution chromatin structure at cis-regulatory elements (CREs). Using C-terminally biotinylated dCas9, endogenous biotin ligases, and pooled sgRNAs, we describe the dCas9-based CAPTURE method for multiplexed analysis of locus-specific chromatin interactions. The redesigned system allows for quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling comparisons across CREs within and between gene clusters. Multiplexed analyses of the spatiotemporal configuration of erythroid super-enhancers and promoter-centric interactions reveal organizational principles of genome structure and function.


Assuntos
Proteína 9 Associada à CRISPR , Cromatina/química , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Biotinilação , Diferenciação Celular/genética , Células Eritroides , Loci Gênicos , Região de Controle de Locus Gênico , Globinas beta/genética
16.
Nature ; 578(7794): 278-283, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025033

RESUMO

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Imagem Molecular , Animais , Remodelação Óssea , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Genes Reporter , Hipóxia/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Masculino , Camundongos , Oxigênio/metabolismo , Crânio/citologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
17.
Nat Genet ; 52(2): 138-145, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959994

RESUMO

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and ß-thalassemia1. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development2-7. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.


Assuntos
Hemoglobinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Adulto , Animais , Sítios de Ligação , Células Cultivadas , Células Eritroides/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica , Hemoglobinas/genética , Humanos , Recém-Nascido , MicroRNAs/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
18.
J Biol Chem ; 295(51): 17738-17751, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33454011

RESUMO

Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Endoderma/citologia , Endoderma/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Histona Desacetilase 1/química , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/química , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3/química , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo
19.
Genome Biol ; 20(1): 192, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500663

RESUMO

We introduce CUT&RUNTools as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short-read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. CUT&RUNTools is available at https://bitbucket.org/qzhudfci/cutruntools/ .


Assuntos
Pegadas de Proteínas/métodos , Software , Cromatina/metabolismo , Endonucleases , Fator de Transcrição GATA1/análise , Humanos , Alinhamento de Sequência , Análise de Sequência de DNA , Células-Tronco/metabolismo
20.
Nature ; 572(7771): 676-680, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391581

RESUMO

The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements1,2. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation3,4 or DNA hypermethylation5 results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)6, is overexpressed in several cancers7-9, but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma10 cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Terapia de Alvo Molecular , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Fenótipo , Ligação Proteica
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