Double PHD Finger Research Articles

In-depth investigation of the effect of pH on the autofluorescence properties of DPF3b and DPF3a amyloid fibrils

Double PHD fingers 3 (DPF3) protein exists as two splicing variants, DPF3b and DPF3a, the involvement of which in human cancer and neurodegeneration is beginning to be increasingly recognised. Both isoforms have recently been identified as intrinsically disordered proteins able to undergo amyloid fibrillation. Upon their aggregation, DPF3 proteins exhibit an intrinsic fluorescence in the visible range, referred to as deep-blue autofluorescence (dbAF). Comprehension of such phenomenon remaining elusive, we investigated in the present study the influence of pH on the optical properties of DPF3b and DPF3a fibrils. By varying the excitation wavelength and the pH condition, the two isoforms were revealed to display several autofluorescence modes that were defined as violet, deep-blue, and blue-green according to their emission range. Complementarily, analysis of excitation spectra and red edge shift plots allowed to better decipher their photoselection mechanism and to highlight isoform-specific excitation-emission features. Furthermore, the observed violation to Kasha-Vavilov’s rule was attributed to red edge excitation shift effects, which were impacted by pH-mediated H-bond disruption, leading to changes in intramolecular charge and proton transfer, or π-electrons delocalisation. Finally, emergence of different autofluorescence emitters was likely related to structurally distinct fibrillar assemblies between isoforms, as well as to discrepancies in the amino acid composition of their aggregation prone regions.

The SWI/SNF chromatin-remodeling subunit DPF2 facilitates NRF2-dependent antiinflammatory and antioxidant gene expression.

During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation, which can cause life-threatening diseases and cancer. Here, we identify a role of double PHD fingers 2 (DPF2) in modulating inflammation. DPF2 is a defining subunit of the hematopoiesis-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncovered that hematopoiesis-specific Dpf2-KO mice developed leukopenia, severe anemia, and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration resembling a clinical hyperinflammatory state. Dpf2 loss impaired the polarization of macrophages responsible for tissue repair, induced the unrestrained activation of Th cells, and generated an emergency-like state of HSC hyperproliferation and myeloid cell-biased differentiation. Mechanistically, Dpf2 deficiency resulted in the loss of the BAF catalytic subunit BRG1 from nuclear factor erythroid 2-like 2-controlled (NRF2-controlled) enhancers, impairing the antioxidant and antiinflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of NRF2 suppressed the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes an essential role of the DPF2-BAF complex in licensing NRF2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.

Abstract 3563: SWI/SNF-associated DPF1 is a unique transcriptional regulator of malignant peripheral nerve sheath tumors

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are typically therapy resistant and associated with a poor prognosis. Genetic aberrations of the polycomb repressive complex 2 (PRC2) occur in up to 75% of MPNST cases. PRC2 functions antagonistically to SWI/SNF protein complexes, regulating expression of target loci in a dynamic relationship that is critical to normal cellular development and maintenance of cell identity. The consequences of PRC2 loss on chromatin state maintained by SWI/SNF in MPNST is unexplored. Therefore, this study used functional genomics to elucidate the role of SWI/SNF in MPNST. Methods: To define the transcriptional regulatory role of SWI/SNF complexes in MPNST, SWI/SNF components were targeted via a CRISPR knock-out (KO) screen combined with a single cell RNA sequencing (RNAseq) readout. Genes of interest highlighted by this screen were further investigated using bulk CRISPR RNAseq. Phenotypic effects of the expression of these genes were studied using loss-of-function assays coupled with colony formation assays. SWI/SNF complex heterogeneity was characterized using glycerol gradient sedimentation, co-immunoprecipitation (co-IP), and mass spectrometry experiments. Results: The investigation of SWI/SNF transcriptional regulation in MPNST highlighted the Double PHD Finger family proteins (DPF1,2,3) as regulating distinct downstream targets. Notably, DPF1 had a unique transcriptional profile compared to other SWI/SNF components. Bulk CRISPR KO RNAseq confirmed these findings and highlighted a specific set of DPF1 target genes including many long non-coding RNAs. The phenotypic role of DPF1 in MPNST was investigated using in vitro siRNA and CRISPR experiments, where DPF1 KO reduced proliferation and viability of MPNST cells in both 2D and 3D cell culture assays. Further, DPF1 was found contribute to anchorage independent cell growth of MPNST cells using soft agar assays. Glycerol gradient sedimentation assays demonstrated that DPF1 co-migrated with core components of a SWI/SNF complex known as canonical BAF (cBAF) and not the alternate GBAF and PBAF SWI/SNF complexes. These findings were further validated using co-immunoprecipitation assays where core the SWI/SNF ATPase, SMARCA4, pulled down DPF1, while components unique to the GBAF and PBAF complexes did not. An ATPase directed proteolysis targeting chimers (PROTAC) was used to therapeutically target cBAF in MPNST cells, reducing their growth and viability. Combination treatment with standard of care chemotherapy synergistically increased the efficacy of this drug. Conclusions: DPF1 was identified as a unique transcriptional regulator of MPNST cells and acts as a member of the cBAF SWI/SNF complex to play important phenotypic roles in this cancer type. Citation Format: Bega Murray, Xiyuan Zhang, Shahroze Abbas, Haiyan Lei, Hilda Jafarah, Jack F. Shern. SWI/SNF-associated DPF1 is a unique transcriptional regulator of malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3563.

Unveiling the Metal-Dependent Aggregation Properties of the C-terminal Region of Amyloidogenic Intrinsically Disordered Protein Isoforms DPF3b and DPF3a

Double-PHD fingers 3 (DPF3) is a BAF-associated human epigenetic regulator, which is increasingly recognised as a major contributor to various pathological contexts, such as cardiac defects, cancer, and neurodegenerative diseases. Recently, we unveiled that its two isoforms (DPF3b and DPF3a) are amyloidogenic intrinsically disordered proteins. DPF3 isoforms differ from their C-terminal region (C-TERb and C-TERa), containing zinc fingers and disordered domains. Herein, we investigated the disorder aggregation properties of C-TER isoforms. In agreement with the predictions, spectroscopy highlighted a lack of a highly ordered structure, especially for C-TERa. Over a few days, both C-TERs were shown to spontaneously assemble into similar antiparallel and parallel β-sheet-rich fibrils. Altered metal homeostasis being a neurodegeneration hallmark, we also assessed the influence of divalent metal cations, namely Cu2+, Mg2+, Ni2+, and Zn2+, on the C-TER aggregation pathway. Circular dichroism revealed that metal binding does not impair the formation of β-sheets, though metal-specific tertiary structure modifications were observed. Through intrinsic and extrinsic fluorescence, we found that metal cations differently affect C-TERb and C-TERa. Cu2+ and Ni2+ have a strong inhibitory effect on the aggregation of both isoforms, whereas Mg2+ impedes C-TERb fibrillation and, on the contrary, enhances that of C-TERa. Upon Zn2+ binding, C-TERb aggregation is also hindered, and the amyloid autofluorescence of C-TERa is remarkably red-shifted. Using electron microscopy, we confirmed that the metal-induced spectral changes are related to the morphological diversity of the aggregates. While metal-treated C-TERb formed breakable and fragmented filaments, C-TERa fibrils retained their flexibility and packing properties in the presence of Mg2+ and Zn2+ cations.

Abstract B011: Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are rare and aggressive soft tissue sarcomas of the peripheral nervous system which are associated with a poor prognosis. Recurrent genetic mutations of the polycomb repressive complex 2 (PRC2) have been identified in up to 75% of MPNST cases. PRC2 is involved in an intricate epigenetic regulatory network in mammalian cells, where it is known to function antagonistically to the SWI/SNF protein complexes which are commonly associated with active transcription. The regulatory relationship between these complexes is critical to normal cell development and maintenance of cellular identity. However, little is known about how PRC2 aberrations affect this regulatory balance in MPNST. Therefore, this study aims to investigate the role of SWI/SNF in MPNST. Methods: A CRISPR knock-out (KO) screen combined with single cell RNA sequencing (RNAseq) was used to target 44 components of SWI/SNF complexes. The transcriptional regulatory effects of resulting genes of interest were further investigated via bulk CRISPR RNAseq experiments, while phenotypic effects were studied using loss-of-function assays coupled with colony formation assays. The dynamic complex formation was further deciphered using glycerol gradient sedimentation and co-immunoprecipitation (co-IP) assays. Results: Double PHD Fingers 1 gene, DPF1, was identified as a unique transcriptional regulator of MPNST via the single cell CRISPR KO screen. These findings were confirmed using a bulk RNAseq screen, where gene set enrichment analysis suggested DPF1 KO has an inhibitory effect on MPNST cell cycle and proliferation through enrichment of genes involved in the P53 pathway (FDR<0.0001) and the apoptotic pathway (FDR<0.0001). This hypothesis was validated using siRNA and CRISPR KO studies in MPNST cell lines, where DPF1 KO led to reduced proliferation and viability of these cells in both 2D and 3D cell culture assays. Soft agar assays were then used to highlight a role of DPF1 to in anchorage independent cell growth of MPNST cells. We then used glycerol gradient sedimentation assays to confirm DPF1 function as a member of the SWI/SNF complexes in MPNST, where DPF1 was observed to co-migrate with SMARCA4, a core ATPase commonly associated with the BAF complex of the SWI/SNF family. This assay was also used to eliminate DPF1 as a member of the GBAF and PBAF SWI/SNF complexes in MPNST, as we identified distinct sedimentation of DPF1 and proteins unique to these complexes. These findings were further validated using co-immunoprecipitation assays, where SMARCA4 had the ability to pull down DPF1, while components of the GBAF and PBAF complexes did not. Conclusions: DPF1 has been identified as a unique regulator of MPNST cells and acts as a member of the BAF SWI/SNF complex. Citation Format: Bega Murray, Xiyuan Zhang, Shahroze Abbas, Haiyan Lei, Hilda Jafarah, Michael C. Kelly, Jack F. Shern. Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B011.

Structural characterisation of amyloidogenic intrinsically disordered zinc finger protein isoforms DPF3b and DPF3a

Double PHD fingers 3 (DPF3) is a zinc finger protein, found in the BAF chromatin remodelling complex, and is involved in the regulation of gene expression. Two DPF3 isoforms have been identified, respectively named DPF3b and DPF3a. Very limited structural information is available for these isoforms, and their specific functionality still remains poorly studied. In a previous work, we have demonstrated the first evidence of DPF3a being a disordered protein sensitive to amyloid fibrillation. Intrinsically disordered proteins (IDPs) lack a defined tertiary structure, existing as a dynamic conformational ensemble, allowing them to act as hubs in protein-protein interaction networks. In the present study, we have more thoroughly characterised DPF3a in vitro behaviour, as well as unravelled and compared the structural properties of the DPF3b isoform, using an array of predictors and biophysical techniques. Predictions, spectroscopy, and dynamic light scattering have revealed a high content in disorder: prevalence of random coil, aromatic residues partially to fully exposed to the solvent, and large hydrodynamic diameters. DPF3a appears to be more disordered than DPF3b, and exhibits more expanded conformations. Furthermore, we have shown that they both time-dependently aggregate into amyloid fibrils, as revealed by typical circular dichroism, deep-blue autofluorescence, and amyloid-dye binding assay fingerprints. Although spectroscopic and microscopic analyses have unveiled that they share a similar aggregation pathway, DPF3a fibrillates at a faster rate, likely through reordering of its C-terminal domain.

A Pilot Study of Whole-Blood Transcriptomic Analysis to Identify Genes Associated with Repetitive Low-Level Blast Exposure in Career Breachers.

Repetitive low-level blast exposure is one of the major occupational health concerns among US military service members and law enforcement. This study seeks to identify gene expression using microRNA and RNA sequencing in whole-blood samples from experienced breachers and unexposed controls. We performed experimental RNA sequencing using Illumina’s HiSeq 2500 Sequencing System, and microRNA analysis using NanoString Technology nCounter miRNA expression panel in whole-blood total RNA samples from 15 experienced breachers and 14 age-, sex-, and race-matched unexposed controls. We identified 10 significantly dysregulated genes between experienced breachers and unexposed controls, with FDR corrected <0.05: One upregulated gene, LINC00996 (long intergenic non-protein coding RNA 996); and nine downregulated genes, IGLV3-16 (immunoglobulin lambda variable 3-16), CD200 (CD200 molecule), LILRB5 (leukocyte immunoglobulin-like receptor B5), ZNF667-AS1 (ZNF667 antisense RNA 1), LMOD1 (leiomodin 1), CNTNAP2 (contactin-associated protein 2), EVPL (envoplakin), DPF3 (double PHD fingers 3), and IGHV4-34 (immunoglobulin heavy variable 4-34). The dysregulated gene expressions reported here have been associated with chronic inflammation and immune response, suggesting that these pathways may relate to the risk of lasting neurological symptoms following high exposures to blast over a career.

The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression

Evolution of clear cell renal cell carcinoma is guided by dysregulation of hypoxia-inducible transcription factor (HIF) pathways following loss of the von Hippel-Lindau tumor suppressor protein. Renal cell carcinoma (RCC)-associated polymorphisms influence HIF–DNA interactions at enhancers of important oncogenes thereby modulating the risk of developing renal cancer. A strong signal of genome-wide association with RCC was determined for the single nucleotide polymorphism (SNP) rs4903064, located on chr14q.24.2 within an intron of DPF3, encoding for Double PHD Fingers 3, a member of chromatin remodeling complexes; however, it is unclear how the risk allele operates in renal cells. In this study, we used tissue specimens and primary renal cells from a large cohort of RCC patients to examine the function of this polymorphism. In clear cell renal cell carcinoma tissue, isolated tumor cells as well as in primary renal tubular cells, in which HIF was stabilized, we determined genotype-specific increases of DPF3 mRNA levels and identified that the risk SNP resides in an active enhancer region, creating a novel HIF-binding motif. We then confirmed allele-specific HIF binding to this locus using chromatin immunoprecipitation of HIF subunits. Consequentially, HIF-mediated DPF3 regulation was dependent on the presence of the risk allele. Finally, we show that DPF3 deletion in proximal tubular cells retarded cell growth, indicating potential roles for DPF3 in cell proliferation. Our analyses suggest that the HIF pathway differentially operates on a SNP-induced hypoxia-response element at 14q24.2, thereby affecting DPF3 expression, which increases the risk of developing renal cancer.

Altered regulation of DPF3, a member of the SWI/SNF complexes, underlies the 14q24 renal cancer susceptibility locus

SummaryOur study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3, a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation.

Revealing Intrinsic Disorder and Aggregation Properties of the DPF3a Zinc Finger Protein.

Double PHD fingers 3 (DPF3) is a human epigenetic factor found in the multiprotein BRG1-associated factor (BAF) chromatin remodeling complex. It has two isoforms: DPF3b and DPF3a, but very little is known about the latter. Despite the lack of structural data, it has been established that DPF3a is involved in various protein–protein interactions and that it is subject to phosphorylation. These features are typical of intrinsically disordered proteins (IDPs) for which the disorder is essential to their functionality. IDPs are also prone to aggregation and can assemble into cytotoxic amyloid fibrils in specific pathological contexts. In the present work, the DPF3a disordered nature and propensity to aggregation have been investigated using a combination of disorder predictors and biophysical methods. The DPF3a-predicted disordered character has been correlated to a characteristic random coil signal in far-UV circular dichroism (CD) and to a fluorescence emission band typical of Trp residues fully exposed to the solvent. After DPF3a purification and 24 h of incubation at room temperature, dynamic light scattering confirmed the presence of DPF3a aggregates whose amyloid nature have been highlighted by a specific deep-blue autofluorescence signature, as well as by an increase in thioflavin T fluorescence upon binding. These results are supported by an enrichment in twisted β-sheets as observed in far-UV CD and a blue shift in intrinsic Trp fluorescence. Both indicate that DPF3a spontaneously tends to orderly aggregate into amyloid fibrils. The diversity of optical signatures originates from dynamical transitions between the disordered and aggregated states of the protein during the incubation. Transmission electron microscopy micrographs reveal that the DPF3a fibrillation process leads to the formation of short needle-shape filaments.

The DPF Domain As a Unique Structural Unit Participating in Transcriptional Activation, Cell Differentiation, and Malignant Transformation.

The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells.

Abstract 401: Functional characterization of the 14q24 renal cancer susceptibility locus implicates SWI/SNF complex member DPF3 via inhibition of apoptosis

Abstract To identify promising regulatory regions under the peaks of Renal Cell Carcinoma (RCC) GWAS signals, we deployed the Massively Parallel Reporter Assay (MPRA), a powerful tool to dissect putative regulatory regions in parallel. Using MPRA, we identified rs4903064 within the 14q24 RCC risk region as a potential transcriptional enhancer with allele-preferential activity. By MPRA, the risk allele C leads to higher expression, an effect confirmed by luciferase assay in three RCC cell lines. Using electromobility shift assays, we observed preferential protein binding to the T allele of rs4903064, one of the strongest signals in the RCC GWAS at 14q24. Motif analysis shows that this SNP resides in a binding site for the transcriptional repressors IRX2 and IRX5 and the C allele is predicted to disrupt the binding site. These results suggest that the higher expression seen in MPRA and luciferase assays when the C allele is present is likely due to reduced binding of transcriptional repressor proteins. Consistent with this, an eQTL analysis of RCC TCGA data showed that C allele is associated with higher expression of the double PHD Fingers 3 gene (DPF3) (p=5.5e-8). DPF3, a plausible candidate gene influenced by the RCC susceptibility allele is a member of the nucleosome remodeling SWI/SNF complex, components of which are frequently mutated in multiple cancers. PBRM1, a member of SWI/SNF complex, is the second most common mutated gene in RCC (40%), demonstrating the importance of SWI/SNF complex for RCC oncogenesis. We generated three kidney cells lines (two RCC- ACHN and UOK-121 and one normal kidney cell line- HK2) with DOX-inducible expression of both DPF3 isoforms (a or b), separately. ATAC-seq analysis of HK2 cells overexpressing DPF3a or DPF3b showed reduced chromatin accessibility compared to controls, suggesting that DPF3 overexpression is disrupting SWI/SNF activity. RNA-seq analysis of DPF3a or DPF3b over expressing cell lines showed deregulation of apoptotic genes, especially overexpression of CEMIP. Cell lines over expressing DPF3a or DPF3b had a higher growth rate, suggesting an oncogenic effect of both DPF3 isoforms. Knockdown of CEMIP by si-RNA blocked the effect of DPF3a and DPF3b on growth rate, suggesting that part of the DPF3 effect on cell growth can be attributed to overexpression of the apoptosis inhibitor CEMIP. We treated cells overexpression of either DPF3a or DPF3b with cisplatin or camptothecin to induce apoptosis and observed cleavage of Caspase 3 and PARP by Western Blot, both markers of apoptosis. Using AnexinV and 7-AAD, flow cytometery analysis confirmed that DPF3a or DPF3b overexpression reduces apoptosis in RCC cell lines. In summary, our results suggest that the RCC risk C-rs4903064 at the 14q24 locus increases expression of DPF3, which disrupts the normal function of the SWI/SNF complex leading to dysregulation of CEMIP and a reduction of apoptosis. Citation Format: Leandro Machado Colli, Lea Jessop, Timothy A. Myers, Mitchell J. Machiela, Jiyeon Choi, Mark Purdue, Kevin Brown, Stephen J. Chanock. Functional characterization of the 14q24 renal cancer susceptibility locus implicates SWI/SNF complex member DPF3 via inhibition of apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 401.

Selective recognition of histone crotonylation by double PHD fingers of MOZ and DPF2.

Recognition of histone covalent modifications by 'reader' modules constitutes a major mechanism for epigenetic regulation. A recent upsurge of newly discovered histone lysine acylations, such as crotonylation (Kcr), butyrylation (Kbu), and propionylation (Kpr), greatly expands the coding potential of histone lysine modifications. Here we demonstrate that the histone acetylation-binding double PHD finger (DPF) domains of human MOZ (also known as KAT6A) and DPF2 (also known as BAF45d) accommodate a wide range of histone lysine acylations with the strongest preference for Kcr. Crystal structures of the DPF domain of MOZ in complex with H3K14cr, H3K14bu, and H3K14pr peptides reveal that these non-acetyl acylations are anchored in a hydrophobic 'dead-end' pocket with selectivity for crotonylation arising from intimate encapsulation and an amide-sensing hydrogen bonding network. Immunofluorescence and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) showed that MOZ and H3K14cr colocalize in a DPF-dependent manner. Our studies call attention to a new regulatory mechanism centered on histone crotonylation readout by DPF family members.

The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification

Histone tail modifications control many nuclear processes by dictating the dynamic exchange of regulatory proteins on chromatin. Here we report novel insights into histone H3 tail structure in complex with the double PHD finger (DPF) of the lysine acetyltransferase MOZ/MYST3/KAT6A. In addition to sampling H3 and H4 modification status, we show that the DPF cooperates with the MYST domain to promote H3K9 and H3K14 acetylation, although not if H3K4 is trimethylated. Four crystal structures of an extended DPF alone and in complex with unmodified or acetylated forms of the H3 tail reveal the molecular basis of crosstalk between H3K4me3 and H3K14ac. We show for the first time that MOZ DPF induces α-helical conformation of H3K4-T11, revealing a unique mode of H3 recognition. The helical structure facilitates sampling of H3K4 methylation status, and proffers H3K9 and other residues for modification. Additionally, we show that a conserved double glycine hinge flanking the H3 tail helix is required for a conformational change enabling docking of H3K14ac with the DPF. In summary, our data provide the first observations of extensive helical structure in a histone tail, revealing the inherent ability of the H3 tail to adopt alternate conformations in complex with chromatin regulators.

Regulation of AML1/RUNX1 Function by Protein Arginine Methyltransferase 4 (PRMT4) in Myeloid Differentiation

Abstract 549RUNX1 (also known as AML1) is the DNA binding component of the Core Binding Factor (CBF)-transcriptional regulatory complex, which plays an important role in hematopoiesis. Upon binding to the common binding sequence -PyGpyGGTPy (Py = pyrimidine) in the regulatory regions of promoters and enhancers of its target genes, RUNX1 acts either as an activator or a repressor, depending on promoter context and its interacting partners. Thus, modulation of the network of RUNX1 interactions can influence hematopoiesis. However, how RUNX1 selects one set of partners over another to assemble a functional complex is largely unknown. Posttranslational modifications, including ubiquitination, phosphorylation, acetylation and methylation, present a viable mean to fine-tune its functions.Here we shown that RUNX1 is arginine methylated at a specific residue, R223, by PRMT4, a type I arginine methyltransferase generally thought of as a co-activator molecule. We hypothesized that arginine methylation of RUNX1 by PRMT4 affects its protein-protein interactions, therefore, to identify proteins that specifically interact with unmethylated and/or methylated-R223 RUNX1, in an unbiased manner, we performed a peptide pull-down experiment, using a methyl-R223 RUNX1 peptide and an unmodified RUNX1 peptide as bait, following by mass spectrometry analysis. We identified several proteins that preferentially interacted with the R223 methyl peptide, but focused on a novel interacting protein, DPF2 (double PhD Finger 2), which is a widely expressed member of the d4 protein family, characterized by the presence of a tandem plant-homodomain (PHD domain). We confirmed the specific interaction between methylated-RUNX1 with DPF2 in vivo by immunoprecipitation. We generated an antibody specific for the R223 methylated-RUNX1 protein, and found that RUNX1 methylation decreases during the myeloid differentiation of human CD34+ haematopoietic stem/progenitor cells (HSPCs), without a change in the total level of RUNX1 protein, and this occurred co-incident with a downregulation of PRMT4 protein expression. Having determined that PRMT4 expression declines during myeloid differentiation, we examined the role of PRMT4 in this process, using short hairpin RNAs to knockdown PRMT4 expression in CD34+ cells. Knockdown of PRMT4 accelerates the myeloid differentiation of the cells, whereas overexpression of PRMT4 in human CD34+ cells blocked their myeloid differentiation. When analyzing the expression of several “master” regulators of myeloid differentiation, we identified microRNA-223, a myeloid specific microRNA, as a common target gene of PRMT4 and RUNX1. Furthermore, we have found that by promoting the assembly of a functional complex containing R223 methylRUNX1 and DPF2 at the transcriptional regulatory region of the microRNA-223 promoter, PRMT4 can control miR-223 expression and myeloid differentiation. We have verified the role of DPF2 in this process, as DPF2 represses miR-223 expression and loss of DPF2 promotes myeloid differentiation. Thus, DPF2 acts in a common pathway with PRMT4 to regulate myeloid differentiation.In conclusion, our study elucidates a novel mechanism, where the arginine methylation of RUNX1 regulates its recruitment of interacting partner(s). In addition to demonstrating that PRMT4 can trigger repression of gene expression, we have identified a novel role for PRMT4 (aka CARM1) in myeloid differentiation. Disclosures:No relevant conflicts of interest to declare.

Double PHD Fingers Protein DPF2 Recognizes Acetylated Histones and Suppresses the Function of Estrogen-related Receptor α through Histone Deacetylase 1

Estrogen-related receptor alpha (ERRalpha) is a member of the nuclear receptor superfamily and regulates many physiological functions, including mitochondrial biogenesis and lipid metabolism. ERRalpha enhances the transactivation function without endogenous ligand by associating with coactivators such as peroxisome proliferator-activated receptor gamma coactivator 1 alpha and beta (PGC-1alpha and -beta) and members of the steroid receptor coactivator family. However, the molecular mechanism by which the transactivation function of ERRalpha is converted from a repressive state to an active state is poorly understood. Here we used biochemical purification techniques to identify ERRalpha-associated proteins in HeLa cells stably expressing ERRalpha. Interestingly, we found that double PHD fingers protein DPF2/BAF45d suppressed PGC-1alpha-dependent transactivation of ERRalpha by recognizing acetylated histone H3 and associating with HDAC1. DPF2 directly bound to ERRalpha and suppressed the transactivation function of nuclear receptors such as androgen receptor. DPF2 was recruited to ERR target gene promoters in myoblast cells, and knockdown of DPF2 derepressed the level of mRNA expressed by target genes of ERRalpha. These results show that DPF2 acts as a nuclear receptor-selective co-repressor for ERRalpha by associating with both acetylated histone H3 and HDAC1.

Regulation of muscle development by DPF3, a novel histone acetylation and methylation reader of the BAF chromatin remodeling complex

Chromatin remodeling and histone modifications facilitate access of transcription factors to DNA by promoting the unwinding and destabilization of histone-DNA interactions. We present DPF3, a new epigenetic key factor for heart and muscle development characterized by a double PHD finger. DPF3 is associated with the BAF chromatin remodeling complex and binds methylated and acetylated lysine residues of histone 3 and 4. Thus, DPF3 may represent the first plant homeodomains that bind acetylated lysines, a feature previously only shown for the bromodomain. During development Dpf3 is expressed in the heart and somites of mouse, chicken, and zebrafish. Morpholino knockdown of dpf3 in zebrafish leads to incomplete cardiac looping and severely reduced ventricular contractility, with disassembled muscular fibers caused by transcriptional deregulation of structural and regulatory proteins. Promoter analysis identified Dpf3 as a novel downstream target of Mef2a. Taken together, DPF3 adds a further layer of complexity to the BAF complex by representing a tissue-specific anchor between histone acetylations as well as methylations and chromatin remodeling. Furthermore, this shows that plant homeodomain proteins play a yet unexplored role in recruiting chromatin remodeling complexes to acetylated histones.