Interplay of BAF and MLL4 promotes cell type-specific enhancer activation.

Cell type-specific enhancers are activated by coordinated actions of lineage-determining transcription factors (LDTFs) and chromatin regulators. The SWI/SNF chromatin remodeling complex BAF and the histone H3K4 methyltransferase MLL4 (KMT2D) are both implicated in enhancer activation. However, the interplay between BAF and MLL4 in enhancer activation remains unclear. Using adipogenesis as a model system, we identify BAF as the major SWI/SNF complex that colocalizes with MLL4 and LDTFs on active enhancers and is required for cell differentiation. In contrast, the promoter enriched SWI/SNF complex PBAF is dispensable for adipogenesis. By depleting BAF subunits SMARCA4 (BRG1) and SMARCB1 (SNF5) as well as MLL4 in cells, we show that BAF and MLL4 reciprocally regulate each other's binding on active enhancers before and during adipogenesis. By focusing on enhancer activation by the adipogenic pioneer transcription factor C/EBPß without inducing cell differentiation, we provide direct evidence for an interdependent relationship between BAF and MLL4 in activating cell type-specific enhancers. Together, these findings reveal a positive feedback between BAF and MLL4 in promoting LDTF-dependent activation of cell type-specific enhancers.

Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2.

Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress.

Eda-activated RelB recruits an SWI/SNF (BAF) chromatin-remodeling complex and initiates gene transcription in skin appendage formation.

Ectodysplasin A (Eda) signaling activates NF-κB during skin appendage formation, but how Eda controls specific gene transcription remains unclear. Here, we find that Eda triggers the formation of an NF-κB-associated SWI/SNF (BAF) complex in which p50/RelB recruits a linker protein, Tfg, that interacts with BAF45d in the BAF complex. We further reveal that Tfg is initially induced by Eda-mediated RelB activation and then bridges RelB and BAF for subsequent gene regulation. The BAF component BAF250a is particularly up-regulated in skin appendages, and epidermal knockout of BAF250a impairs skin appendage development, resulting in phenotypes similar to those of Eda-deficient mouse models. Transcription profiling identifies several target genes regulated by Eda, RelB, and BAF. Notably, RelB and the BAF complex are indispensable for transcription of Eda target genes, and both BAF complex and Eda signaling are required to open chromatin of Eda targets. Our studies thus suggest that Eda initiates a signaling cascade and recruits a BAF complex to specific gene loci to facilitate transcription during organogenesis.

Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks.

The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. Here, we successfully observed recruitment of FANCM at stalled forks using a newly developed protocol. We report that the FANCM recruitment depends upon its intrinsic DNA translocase activity, and its DNA-binding partner FAAP24. Moreover, it is dependent on the replication checkpoint kinase, ATR; but is independent of the FA core and FANCD2-FANCI complexes, two essential components of the FA pathway, indicating that the FANCM recruitment occurs downstream of ATR but upstream of the FA pathway. Interestingly, the recruitment of FANCM requires its direct interaction with Bloom syndrome complex composed of BLM helicase, Topoisomerase 3α, RMI1 and RMI2; as well as the helicase activity of BLM. We further show that the FANCM-BLM complex interaction is critical for replication stress-induced FANCM hyperphosphorylation, for normal activation of the FA pathway in response to ICLs, and for efficient traverse of ICLs by the replication machinery. Epistasis studies demonstrate that FANCM and BLM work in the same pathway to promote replication traverse of ICLs. We conclude that FANCM and BLM complex work together at stalled forks to promote both FA repair and replication traverse pathways of ICLs.

BAF250a Protein Regulates Nucleosome Occupancy and Histone Modifications in Priming Embryonic Stem Cell Differentiation.

The unique chromatin signature of ES cells is fundamental to the pluripotency and differentiation of ES cells. One key feature is the poised chromatin state of master developmental genes that are transcriptionally repressed in ES cells but ready to be activated in response to differentiation signals. Poised chromatin in ES cells contains both H3 Lys-4 trimethylation (H3K4me3) and H3 Lys-27 trimethylation (H3K27me3) methylation, indicating activating and repressing potential. However, the contribution of non-covalent chromatin structure to the poised state is not well understood. To address whether remodeling of nucleosomes is important to the poised state, we characterized the function of BAF250a, a key regulatory subunit of the ES cell ATP-dependent Brahma-associated factor (BAF) chromatin remodeling complex (esBAF). Acute deletion of BAF250a disrupted the differentiation potential of ES cells by altering the expression timing of key developmental genes and pluripotent genes. Our genome-wide nucleosome and histone modification analyses indicated that the disruption of gene expression timing was largely due to changes of chromatin structures at poised genes, particularly those key developmental genes mediated by BAF250a. Specifically, BAF250a deletion caused a nucleosome occupancy increase at H3K4me3- and/or H3K27me3-associated promoters. Moreover, H3K27me3 levels and the number of bivalent promoter genes were reduced in BAF250a KO ES cells. We revealed that BAF250a ablation led to elevated Brg1 but reduced Suz12 recruitment at nucleosome occupancy-increased regions, indicating an unexpected and complicated role of BAF250a in regulating esBAF and Polycomb repressive complex (PRC) activities. Together, our studies identified that BAF250a mediates esBAF and PRC functions to establish the poised chromatin configuration in ES cells, which is essential for the proper differentiation of ES cells.

The histone-fold complex MHF is remodeled by FANCM to recognize branched DNA and protect genome stability.

Histone-fold proteins typically assemble in multiprotein complexes to bind duplex DNA. However, one histone-fold complex, MHF, associates with Fanconi anemia (FA) protein FANCM to form a branched DNA remodeling complex that senses and repairs stalled replication forks and activates FA DNA damage response network. How the FANCM-MHF complex recognizes branched DNA is unclear. Here, we solved the crystal structure of MHF and its complex with the MHF-interaction domain (referred to as MID) of FANCM, and performed structure-guided mutagenesis. We found that the MID-MHF complex consists of one histone H3-H4-like MHF heterotetramer wrapped by a single polypeptide of MID. We identified a zinc atom-liganding structure at the central interface between MID and MHF that is critical for stabilization of the complex. Notably, the DNA-binding surface of MHF was altered by MID in both electrostatic charges and allosteric conformation. This leads to a switch in the DNA-binding preference - from duplex DNA by MHF alone, to branched DNA by the MID-MHF complex. Mutations that disrupt either the composite DNA-binding surface or the protein-protein interface of the MID-MHF complex impaired activation of the FA network and genome stability. Our data provide the structural basis of how FANCM and MHF work together to recognize branched DNA, and suggest a novel mechanism by which histone-fold complexes can be remodeled by their partners to bind special DNA structures generated during DNA metabolism.

A ubiquitin-binding protein, FAAP20, links RNF8-mediated ubiquitination to the Fanconi anemia DNA repair network.

The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.

A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability.

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

BAF250B-associated SWI/SNF chromatin-remodeling complex is required to maintain undifferentiated mouse embryonic stem cells.

Whether SWI/SNF chromatin remodeling complexes play roles in embryonic stem (ES) cells remains unknown. Here we show that SWI/SNF complexes are present in mouse ES cells, and their composition is dynamically regulated upon induction of ES cell differentiation. For example, the SWI/SNF purified from undifferentiated ES cells contains a high level of BAF155 and a low level of BAF170 (both of which are homologs of yeast SWI3 protein), whereas that from differentiated cells contains nearly equal amounts of both. Moreover, the levels of BAF250A and BAF250B decrease during the differentiation of ES cells, whereas that of BRM increases. The altered expression of SWI/SNF components hinted that these complexes could play roles in ES cell maintenance or differentiation. We therefore generated ES cells with biallelic inactivation of BAF250B and found that these cells display a reduced proliferation rate and an abnormal cell cycle. Importantly, these cells are deficient in the self-renewal capacity of undifferentiated ES cells and exhibit certain phenotypes of differentiated cells, including reduced expression of several pluripotency-related genes and increased expression of some differentiation-related genes. These data suggest that the BAF250B-associated SWI/SNF is essential for mouse ES cells to maintain their normal proliferation and pluripotency. The work presented here underscores the importance of SWI/SNF chromatin remodeling complexes in pluripotent stem cells.

FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway.

The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100-deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA-BRCA DNA damage response network.

Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM.

The Fanconi anemia (FA) core complex plays a crucial role in a DNA damage response network with BRCA1 and BRCA2. How this complex interacts with damaged DNA is unknown, as only the FA core protein FANCM (the homolog of an archaeal helicase/nuclease known as HEF) exhibits DNA binding activity. Here, we describe the identification of FAAP24, a protein that targets FANCM to structures that mimic intermediates formed during the replication/repair of damaged DNA. FAAP24 shares homology with the XPF family of flap/fork endonucleases, associates with the C-terminal region of FANCM, and is a component of the FA core complex. FAAP24 is required for normal levels of FANCD2 monoubiquitylation following DNA damage. Depletion of FAAP24 by siRNA results in cellular hypersensitivity to DNA crosslinking agents and chromosomal instability. Our data indicate that the FANCM/FAAP24 complex may play a key role in recruitment of the FA core complex to damaged DNA.

PBAF chromatin-remodeling complex requires a novel specificity subunit, BAF200, to regulate expression of selective interferon-responsive genes.

PBAF and BAF are two chromatin-remodeling complexes of the SWI/SNF family essential for mammalian transcription and development. Although these complexes share eight identical subunits, only PBAF can facilitate transcriptional activation by nuclear receptors in vitro. Here we show that these complexes have selectivity in mediating transcription of different interferon-responsive genes. The selectivity by PBAF requires a novel subunit, BAF200, but not the previously described PBAF-specificity subunit, BAF180 (Polybromo). Our study provides in vivo evidence that PBAF and BAF regulate expression of distinct genes, and suggests that BAF200 plays a key role in PBAF function.

Loss of the INI1 tumor suppressor does not impair the expression of multiple BRG1-dependent genes or the assembly of SWI/SNF enzymes.

The INI1/hSNF5 tumor suppressor is an integral component of mammalian SWI/SNF chromatin remodeling enzymes that contain SNF2 family ATPases BRM (Brahma) or BRG1 (Brahma Related Gene 1) and that contribute to the regulation of many genes. Genetic studies of yeast SWI/SNF enzyme revealed similar phenotypes when single or multiple components of the enzyme were deleted, indicating a requirement for each subunit. To address the contribution of INI1 in the regulation of SWI/SNF-dependent genes in mammalian cells, we examined the expression of multiple BRG1-dependent, constitutively expressed genes in INI1-deficient cancer cell lines. At least one INI1-deficient line expressed each gene, and reintroduction of INI1 had negligible effects on expression levels. Lack of INI1 also did not prevent interferon gamma (IFNgamma)-mediated induction of CIITA, which is BRG1 dependent, and GBP-1, which is BRG1 enhanced, and reintroduction of INI1 had minimal effects. Chromatin immunoprecipitation experiments revealed that BRG1 inducibly binds to the CIITA promoter despite the absence of INI1. Unlike yeast deleted for the INI1 homologue, SWI/SNF enzymes in INI1-deficient cells were largely intact. Thus in human cells, SWI/SNF enzyme complex formation and the expression of many BRG1-dependent genes are independent of INI1.

FANCL replaces BRCA1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination.

Monoubiquitination of FANCD2 is a key step in the DNA damage response pathway involving Fanconi anemia proteins and the breast cancer susceptibility gene products, BRCA1 and BRCA2. One critical unresolved issue is the identity of the ubiquitin ligase responsible for this reaction. Two proteins, BRCA1 and FANCL(PHF9), have been suggested to be this ligase. Here we found that FANCL, but not BRCA1, evolutionarily co-exists with FANCD2 in several species. Moreover, the proportion of FANCD2 in chromatin and nuclear matrix is drastically reduced in a cell line mutated in FANCL, but not in that mutated in BRCA1. This defective distribution of FANCD2 in the FANCL-mutant cell line is likely due to its defective monoubiquitination, because the monoubiquitinated FANCD2 preferentially associates with chromatin and nuclear matrix, whereas non-ubiquitinated FANCD2 largely resides in the soluble fraction. Our data support the notion that FANCL, but not BRCA1, is the likely ligase for FANCD2 monoubiquitination.

Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1.

Understanding molecular mechanisms regulating angiogenesis may lead to novel therapies for ischemic disorders. Hypoxia-inducible factor 1 (HIF-1) activates vascular endothelial growth factor (VEGF) gene expression in hypoxic/ischemic tissue. In this study we demonstrate that exposure of primary cultures of cardiac and vascular cells to hypoxia or AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, modulates the expression of genes encoding the angiogenic factors angiopoietin-1 (ANGPT1), ANGPT2, placental growth factor, and platelet-derived growth factor-B. Loss-of-function effects were also observed in HIF-1alpha-null embryonic stem cells. Depending on the cell type, expression of ANGPT1 and ANGPT2 was either activated or repressed in response to hypoxia or AdCA5. In all cases, there was complete concordance between the effects of hypoxia and AdCA5. Injection of AdCA5 into mouse eyes induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Analysis of gene expression revealed increased expression of ANGPT1, ANGPT2, platelet-derived growth factor-B, placental growth factor, and VEGF mRNA in AdCA5-injected eyes. These results indicate that HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors and that adenovirus-mediated expression of a constitutively active form of HIF-1alpha is sufficient to induce angiogenesis in nonischemic tissue of an adult animal.

A novel ubiquitin ligase is deficient in Fanconi anemia.

Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.

ATRX syndrome is characterized by X-linked mental retardation associated with alpha-thalassemia. The gene mutated in this disease, ATRX, encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: (i) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; (ii) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells of a patient with ATRX syndrome, the level of the Daxx-ATRX complex is correspondingly reduced; (iv) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and (v) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

Novel SWI/SNF chromatin-remodeling complexes contain a mixed-lineage leukemia chromosomal translocation partner.

The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

RAP80, a novel nuclear protein that interacts with the retinoid-related testis-associated receptor.

In this study, we describe the characterization of a novel nuclear protein, referred to as RAP80. The RAP80 cDNA was cloned from a human testis cDNA library and encodes a 719-amino acid protein containing two potential CX(2)CX(11)HX(3)C-type zinc finger motifs at its carboxyl-terminal region. Analysis of its genomic structure revealed that the RAP80 gene covers more than 90 kb and consists of 15 exons and 14 introns. Fluorescence in situ hybridization mapped the RAP80 gene to human chromosome 5q35. RAP80 mRNA is expressed in many human tissues, but its expression is particularly high in testis. In situ hybridization showed that RAP80 is highly expressed in germ cells of mouse testis but is not differentially regulated during spermatogenesis. Confocal microscopy showed that RAP80 is localized to the nucleus, where it is distributed in a speckled pattern. Deletion analysis showed that a bipartite nuclear localization signal at the amino terminus is important in mediating nuclear transport of RAP80. Monohybrid analysis showed that RAP80 might function as an active repressor of transcription. Mammalian two-hybrid analysis demonstrated that RAP80 was able to interact with the retinoid-related testis-associated receptor (RTR), an orphan receptor that has been implicated in the control of embryonic development and spermatogenesis. Pull-down analysis showed that RAP80 and RTR physically interact in vitro. Deletion and point mutation analyses revealed that part of the hinge domain of RTR is required for this interaction. RAP80 is able to inhibit the interaction of RTR with the co-repressor N-CoR likely by competing with N-CoR for RTR binding. Our results suggest that RAP80 may be functioning as a modulator of RTR signaling.