C-terminal Plant Homeodomain Research Articles

Molecular basis for PHF7-mediated ubiquitination of histone H3.

The RING-type E3 ligase has been known for over two decades, yet its diverse modes of action are still the subject of active research. Plant homeodomain (PHD) finger protein 7 (PHF7) is a RING-type E3 ubiquitin ligase responsible for histone ubiquitination. PHF7 comprises three zinc finger domains: an extended PHD (ePHD), a RING domain, and a PHD. While the function of the RING domain is largely understood, the roles of the other two domains in E3 ligase activity remain elusive. Here, we present the crystal structure of PHF7 in complex with the E2 ubiquitin-conjugating enzyme (E2). Our structure shows that E2 is effectively captured between the RING domain and the C-terminal PHD, facilitating E2 recruitment through direct contact. In addition, through in vitro binding and functional assays, we demonstrate that the N-terminal ePHD recognizes the nucleosome via DNA binding, whereas the C-terminal PHD is involved in histone H3 recognition. Our results provide a molecular basis for the E3 ligase activity of PHF7 and uncover the specific yet collaborative contributions of each domain to the PHF7 ubiquitination activity.

Novel insights into the recognition of acetylated histone H4 tail by the TRIM24 PHD-Bromo module.

TRIM24 is a multi-functional chromatin reader, and it binds to the estrogen receptor to activate estrogen-dependent target genes associated with tumor development. TRIM24 is known to ubiquitinate p53 via an N-terminal RING domain and binds a specific combinatorial histone signature of H3K4me0/H3K23ac via its C-terminal plant homeodomain (PHD) and bromodomain (Bromo). Aberrant expression of TRIM24 positively correlates with H3K23ac levels, and high levels of both TRIM24 and H3K23ac predict poor survival of breast cancer patients. Little has been explored about the acetylated histone H4 (H4ac) signatures of TRIM24 and their biological functions. Herein, we report novel H4ac binding partners of TRIM24 and their localization in the genome. Isothermal titration calorimetry binding assay on the histone peptides revealed that the TRIM24 PHD-Bromo preferably binds to H4K5ac, H4K8ac, and H4K5acK8ac compared with other acetylated histone H4 ligands. Co-immunoprecipitation on the endogenous histones suggests that the recognition of H4ac by Bromo does not interfere with the recognition of H3K4me0 mark by the PHD domain of TRIM24. Consistent with this, TRIM24 PHD-Bromo exhibits minimal discrimination among H4ac binding partners at endogenous histone and nucleosome levels. Moreover, ChIP-seq analysis revealed that the H4K5ac and H4K8ac histone signatures strongly co-localize near the transcription start sites of different hub genes or TRIM24-targeted genes in breast cancer. In addition, the KEGG pathway analysis demonstrates that the TRIM24 and its H4ac targets are associated with several important biological pathways. Our findings describe that the H4ac recognition by TRIM24 PHD-Bromo enables access to the chromatin for specific transcriptional regulation.

Identification of Histone Peptide Binding Specificity and Small-Molecule Ligands for the TRIM33α and TRIM33β Bromodomains.

TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1β), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33β, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33β, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31–27K18Ac, and bind preferentially to H31–27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.

Characterisation of class VI TRIM RING domains: linking RING activity to C-terminal domain identity.

TRIM E3 ubiquitin ligases regulate multiple cellular processes, and their dysfunction is linked to disease. They are characterised by a conserved N-terminal tripartite motif comprising a RING, B-box domains, and a coiled-coil region, with C-terminal domains often mediating substrate recruitment. TRIM proteins are grouped into 11 classes based on C-terminal domain identity. Class VI TRIMs, TRIM24, TRIM33, and TRIM28, have been described as transcriptional regulators, a function linked to their C-terminal plant homeodomain and bromodomain, and independent of their ubiquitination activity. It is unclear whether E3 ligase activity is regulated in family members where the C-terminal domains function independently. Here, we provide a detailed biochemical characterisation of the RING domains of class VI TRIMs and describe the solution structure of the TRIM28 RING. Our study reveals a lack of activity of the isolated RING domains, which may be linked to the absence of self-association. We propose that class VI TRIMs exist in an inactive state and require additional regulatory events to stimulate E3 ligase activity, ensuring that associated chromatin-remodelling factors are not injudiciously degraded.

Tripartite Motif Cofactors, a Novel Gene Target for Liver Cancer via Regulating the Immune Cells and Gut Microbiome: A Review

TRIM, a multi-domain protein associated with N-terminal ring finger E3 ligase and C-terminal plant homeodomain/ bromodomain PHD chromatin interacting module, N-terminal ring finger known as ring B-boxes and coiled-coil RBCC domain with structure that underscores biochemical reaction which requires enzymes like E1, E2 and E3 of which E3 serves as receptor recognition for target proteins. Most TRIM proteins are E3 ligases in the ubiquitination cascade that translated in diverse physiological and biological processes such as differentiation, growth, transcription and oncogenesis. Implicated in pathological processes from Mendelian inherited disorders, cellular (plethora) processes like cell cycle regulation, innate immune response and apoptosis to cancer. Genetic factors are at high risk and contributed between 30%-50% disease prevalence like obesity, cirrhosis. TRIM28 (TIF1β), TRIM24 (TIF1α), TRIM33 (TIF1γ) are cofactors of tripartite motif TRIM subfamily protein, distinct transcriptional factors that correlate with each other and interact with other proteins both in functional and physical in cancer disease. Studies have shown that TRIM protein is a regulator in inflammatory, infectious and cancer diseases. This review focused on tripartite genes as a liver cancer target via regulating immune cells and the gut microbiome. More on research so far, disease development, progression and influence. Considering the incident rate and progression, genetic involvement remain challenging thus needs more insight on prognosis that will potentiate clinical effect with lesser adverse events and recurrences that will benefit the patients.

PHD finger protein 1 (PHF1) is a novel reader for histone H4R3 symmetric dimethylation and coordinates with PRMT5-WDR77/CRL4B complex to promote tumorigenesis.

Histone post–translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains. PHF1 [plant homeodomain (PHD) finger protein 1], which contains two kinds of histone reader modules, a Tudor domain and two PHD fingers, is an essential factor for epigenetic regulation and genome maintenance. While significant progress has been made in characterizing the function of the Tudor domain, the roles of the two PHD fingers are poorly defined. Here, we demonstrated that the N-terminal PHD finger of PHF1 recognizes symmetric dimethylation of H4R3 (H4R3me2s) catalyzed by PRMT5–WDR77. However, the C-terminal PHD finger of PHF1, instead of binding to modified histones, directly interacts with DDB1, the main component of the CUL4B-Ring E3 ligase complex (CRL4B), which is responsible for H2AK119 mono-ubiquitination (H2AK119ub1). We showed that PHF1, PRMT5–WDR77, and CRL4B reciprocally interact with one another and collaborate as a functional unit. Genome-wide analysis of PHF1/PRMT5/CUL4B targets identified a cohort of genes including E-cadherin and FBXW7, which are critically involved in cell growth and migration. We demonstrated that PHF1 promotes cell proliferation, invasion, and tumorigenesis in vivo and in vitro and found that its expression is markedly upregulated in a variety of human cancers. Our data identified a new reader for H4R3me2s and provided a molecular basis for the functional interplay between histone arginine methylation and ubiquitination. The results also indicated that PHF1 is a key factor in cancer progression, supporting the pursuit of PHF1 as a target for cancer therapy.

Abstract 110: Molecular profiling of Tudor domain-containing proteins identifies PHF20L1 as a candidate oncogene in breast cancer

Abstract Tudor domain-containing proteins (TDRDs), which recognize and bind to methyl-lysine/arginine residues on histones and non-histone proteins, play critical roles in regulating chromatin architecture, transcription, genomic stability, and RNA metabolism. Dysregulation of several TDRDs have been observed in various types of cancer. However, neither the genomic landscape nor clinical significance of TDRDs in breast cancer has been explored comprehensively. Here, we performed an integrated genomic and transcriptomic analysis of 41 TDRD genes in breast cancer (TCGA and METABRIC datasets) and identified associations among recurrent copy number alterations, gene expressions, clinicopathological features, and survival of patients. Among seven TDRDs (PHF20L1, ARID4B, SETDB1, LBR, TDRKH, TDRD10, and TDRD5) that had the highest frequency (>10%) of gene amplification, the plant homeodomain finger protein 20-like 1 (PHF20L1) was the most commonly amplified (17.62%) TDRD gene in TCGA breast cancers. Different subtypes of breast cancer had different patterns of copy number and expression for each TDRD. Notably, amplification and overexpression of PHF20L1 were more prevalent in aggressive basal-like and Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. Furthermore, knockdown of PHF20L1 inhibited cell proliferation in PHF20L1-amplified breast cancer cell lines. PHF20L1 protein contains N-terminal Tudor and C-terminal plant homeodomain domains. Detailed characterization of PHF20L1 in breast cancer revealed that the Tudor domain likely plays a critical role in promoting cancer. Mechanistically, PHF20L1 might participate in regulating DNA methylation by stabilizing DNA methyltransferase 1 (DNMT1) protein in breast cancer. Thus, our results demonstrated the oncogenic potential of PHF20L1 and its association with poor prognostic parameters in breast cancer. Furthermore, our findings provide strong foundations for further research and therapeutic targeting of PHF20L1 or other TDRDs in breast cancer. Citation Format: Yuanyuan Jiang, Lanxin Liu, Wenqi Shan, Zeng-Quan Yang. Molecular profiling of Tudor domain-containing proteins identifies PHF20L1 as a candidate oncogene in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 110.

An integrated genomic analysis of Tudor domain–containing proteins identifies PHD finger protein 20-like 1 (PHF20L1) as a candidate oncogene in breast cancer

Tudor domain–containing proteins (TDRDs), which recognize and bind to methyl-lysine/arginine residues on histones and non-histone proteins, play critical roles in regulating chromatin architecture, transcription, genomic stability, and RNA metabolism. Dysregulation of several TDRDs have been observed in various types of cancer. However, neither the genomic landscape nor clinical significance of TDRDs in breast cancer has been explored comprehensively. Here, we performed an integrated genomic and transcriptomic analysis of 41 TDRD genes in breast cancer (TCGA and METABRIC datasets) and identified associations among recurrent copy number alterations, gene expressions, clinicopathological features, and survival of patients. Among seven TDRDs that had the highest frequency (>10%) of gene amplification, the plant homeodomain finger protein 20-like 1 (PHF20L1) was the most commonly amplified (17.62%) TDRD gene in TCGA breast cancers. Different subtypes of breast cancer had different patterns of copy number and expression for each TDRD. Notably, amplification and overexpression of PHF20L1 were more prevalent in aggressive basal-like and Luminal B subtypes and were significantly associated with shorter survival of breast cancer patients. Furthermore, knockdown of PHF20L1 inhibited cell proliferation in PHF20L1-amplified breast cancer cell lines. PHF20L1 protein contains N-terminal Tudor and C-terminal plant homeodomain domains. Detailed characterization of PHF20L1 in breast cancer revealed that the Tudor domain likely plays a critical role in promoting cancer. Mechanistically, PHF20L1 might participate in regulating DNA methylation by stabilizing DNA methyltransferase 1 (DNMT1) protein in breast cancer. Thus, our results demonstrated the oncogenic potential of PHF20L1 and its association with poor prognostic parameters in breast cancer.

Expression of inhibitor of growth 4 by HSV1716 improves oncolytic potency and enhances efficacy

We have isolated and characterized a novel variant of the replication-competent oncolytic HSV1716 that expresses inhibitor of growth 4 (Ing4) (HSV1716Ing4). We demonstrate that Ing4 expression enhances progeny output during HSV1716 infection of human tumor cells both in vitro and in vivo, thereby significantly augmenting its oncolytic potency. In tissue culture, compared with HSV1716, HSV1716Ing4 produced significantly higher numbers of infectious progeny in human squamous cell carcinoma (SCC), breast, ovarian, prostate and colorectal cancer cell lines. Immediate-early expression of Ing4 was crucial for this effect and an intact Ing4 was required as there was no enhanced progeny production with HSV1716 variants that expressed Ing4 mutants lacking the C-terminal plant homeodomain domain or conserved nuclear localization signals. In mouse xenograft models of SCC, ovarian and breast cancer, HSV1716Ing4 was significantly more efficacious than HSV1716 with at least 1000-fold more infectious virus found in tumors after HSV1716Ing4 treatment compared with tumors from HSV1716 treatment. Using a sensitive herpes simplex virus type 1 (HSV-1) PCR, virus DNA was only detected in tumors and was not detected in the DNA extracted from any organs of the injected mice demonstrating that, like HSV1716, HSV1716Ing4 replication is exclusively restricted to tumor cells. Our results suggest that the potential for enhanced tumor destruction by oncolytic HSV expressing Ing4 merits clinical investigation.

Crystal Structure of Inhibitor of Growth 4 (ING4) Dimerization Domain Reveals Functional Organization of ING Family of Chromatin-binding Proteins

The protein ING4 binds to histone H3 trimethylated at Lys-4 (H3K4me3) through its C-terminal plant homeodomain, thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the plant homeodomain finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure. This arrangement suggests that ING4 can bind simultaneously two histone tails on the same or different nucleosomes. Dimerization has a direct impact on ING4 tumor suppressor activity because monomeric mutants lose the ability to induce apoptosis after genotoxic stress. Homology modeling based on the ING4 structure suggests that other ING dimers may also exist.

ASXL1 Mutations Predict for Resistance to Chemotherapy and Inferior Outcome in Younger Adult Patients with Acute Myeloid Leukemia (AML): A Study of the German-Austrian AMLSG

Abstract 412 Background:Somatic mutations of the ASXL1 (Additional Sex Comb-Like 1) gene on chromosome 20q11.1 were identified in various myeloid malignancies with the highest incidence reported in CMML (∼40%) and lower frequencies in MDS, AML, CML, and myeloproliferative neoplasia. The ASXL1 protein has been suggested to act as chromatin modifier and the highly conserved C-terminal plant homeo-domain (PHD) finger is presumably critical for its function. ASXL1 mutations cluster in exon 12 and are mainly frameshift mutations predicted to remove the PHD domain. We and others (Paschka et al., Haematologica 2011;96(s2);425; Chou et al., Blood 2010;116:4086–94) have recently reported first results on the unfavorable prognostic impact of ASXL1 mutations in AML. However, the clinical relevance of these mutations still needs to be elucidated in larger AML cohorts. Methods:Mutational analyses of ASXL1 were performed on diagnostic samples from 1429 patients with AML aged 18 to 61 years. All patients were intensively treated on one of two AMLSG trials [AML HD98A (n=745), Schlenk et al., J Clin Oncol. 2010;28:4642–8; AMLSG 07–04(n=684), NCT00151242]. GeneScan-based fragment analysis of several amplicons spanning ASXL1 exon 12 was used to screen for ASXL1 mutations. Samples showing altered GeneScan profiles were amplified in a second PCR reaction, and the amplicons were sequenced to confirm the mutation and to determine the mutation type. Patients were also assessed for the presence of NPM1, FLT3 (ITD and TKD), CEBPA, IDH1/2, RUNX1, and DNMT3A mutations by standard PCR-based methods. Results:ASXL1 mutations were detected in 90 (5.9%) of 1429 patients. All mutations were heterozygous frameshifts predicted to cause loss of the PHD finger. The most common mutation was a duplication of guanine at position 1934 (c.1934dup) identified in 59% (53/90) of the mutated cases. Three other ASXL1 mutations were detected in more than one patient: c.1900_1922del (n=16), c.1934del (n=5), c.1960dup (n=3). The majority of mutations (91%) clustered within or around a glycine-rich protein domain spanning amino acids 642–685. Patients with ASXL1 mutations were older (P<.0001), more frequently males (P=.03), and they more frequently had secondary AML (P=.02). They also showed lower values for WBC (P=.01), bone marrow (P=.0002) and circulating (P=.001) blasts, and LDH serum levels (P=.007). ASXL1 mutations were more frequent in patients exhibiting intermediate-risk cytogenetics (P=.06). Among the ASXL1 mutated cases, 42% were cytogenetically normal (CN), 23% had other intermediate-risk cytogenetics, 26% had high-risk, and 9% low-risk [t(8;21) n=7, t(15;17) n=1, no inv(16)/t(16;16)] cytogenetics. ASXL1 mutations were associated with RUNX1 (P<.001) and IDH2R140 mutations (P=.006). In contrast, ASXL1 mutations were less frequent in patients with NPM1 (P<.001), FLT3-ITD (P<.001), and DNMT3A (P=.02) mutations. The median follow-up for survival was 4.8 years. Patients with an ASXL1 mutation had an inferior complete remission (CR) rate compared with ASXL1 wildtype patients (57% vs 74%; P<.001); the same was true for the subset of CN-AML (58% vs 77%; P=.04). In both, the entire cohort (P=.04) and in CN-AML (P=.07) ASXL1 mutations were associated with a worse event-free-survival, whereas no impact of these mutations was observed on relapse-free-survival. Patients with ASXL1 mutation had a shorter overall survival (OS) compared to those with ASXL1 wildtype (P=.002; 4-year OS rates, 32% vs 47%). The adverse impact of ASXL1 mutations on OS was also present in the subgroup of CN-AML (P=.009; 4-year OS rates, 33% vs 48%). In multivariable analysis, ASXL1 mutation was in trend an unfavorable factor for OS in CN-AML (hazard ratio: 1.46; P=.087). Conclusions:ASXL1 mutations were identified in ∼6% of younger AML patients and were associated with intermediate-risk cytogenetics. ASXL1 mutations frequently occurred with RUNX1 and IDH2R140 mutations, and were less frequent in NPM1 and FLT3-ITD mutated AML. ASXL1 mutations were associated with lower CR rate and inferior OS. The exact role of ASXL1 in normal hematopoiesis still needs to be defined in functional studies as well as the potential role of mutated ASXL1 protein in mediating resistance to chemotherapy. Disclosures:Kündgen:Celgene: Honoraria; Novartis: Honoraria.

Activity of a C-terminal Plant Homeodomain (PHD) of Msc1 Is Essential for Function

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin. To assess the function of Msc1 in the Swr1 complex, domains of Msc1 necessary for interaction with Swr1 were identified. The C-terminal plant homeodomain (PHD) 2 and PHD3 of Msc1 are sufficient to confer association with Swr1 and allow Msc1 to function in the context of kinetochore mutants. On the other hand, a mutant with a single amino acid substitution in PHD2 within the full-length Msc1 protein retains the ability to bind to Swr1 but eliminates the function of Msc1 in combination with kinetochore mutants. Thus, Swr1 association is critical but not sufficient for Msc1 function. An activity of Msc1 that depends on the cysteine residue within PHD2 of Msc1 is likewise critical for function. On the basis of our observation that the PHDs of Msc1 act as E3 ubiquitin ligases and that mutations of cysteine residues within those domains abolish ligase activity, we speculate that the ability of Msc1 to facilitate ubiquitin transfer is critical for the function it mediates through its association with Swr1.

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Critical Determinants for Chromatin Binding by Saccharomyces cerevisiae Yng1 Exist Outside of the Plant Homeodomain Finger

The temporal and spatial regulation of histone post-translational modifications is essential for proper chromatin structure and function. The Saccharomyces cerevisiae NuA3 histone acetyltransferase complex modifies the amino-terminal tail of histone H3, but how NuA3 is targeted to specific regions of the genome is not fully understood. Yng1, a subunit of NuA3 and a member of the Inhibitor of Growth (ING) protein family, is required for the interaction of NuA3 with chromatin. This protein contains a C-terminal plant homeodomain (PHD) finger that specifically interacts with lysine 4-trimethylated histone H3 (H3K4me3) in vitro. This initially suggested that NuA3 is targeted to regions bearing the H3K4me3 mark; however, deletion of the Yng1 PHD finger does not disrupt the interaction of NuA3 with chromatin or result in a phenotype consistent with loss of NuA3 function in vivo. In this study, we uncovered the molecular basis for the discrepancies in these data. We present both genetic and biochemical evidence that full-length Yng1 has two independent histone-binding motifs: an amino-terminal motif that binds unmodified H3 tails and a carboxyl-terminal PHD finger that specifically recognizes H3K4me3. Although these motifs can bind histones independently, together they increase the apparent association of Yng1 for the H3 tail.

The Fission Yeast Inhibitor of Growth (ING) Protein Png1p Functions in Response to DNA Damage

In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

Loss-of-function Additional sex combs like 1 mutations disrupt hematopoiesis but do not cause severe myelodysplasia or leukemia

AbstractThe Additional sex combs like 1 (Asxl1) gene is 1 of 3 mammalian homologs of the Additional sex combs (Asx) gene of Drosophila. Asx is unusual because it is required to maintain both activation and silencing of Hox genes in flies and mice. Asxl proteins are characterized by an amino terminal homology domain, by interaction domains for nuclear receptors, and by a C-terminal plant homeodomain protein-protein interaction domain. A recent study of patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) revealed a high incidence of truncation mutations that would delete the PHD domain of ASXL1. Here, we show that Asxl1 is expressed in all hematopoietic cell fractions analyzed. Asxl1 knockout mice exhibit defects in frequency of differentiation of lymphoid and myeloid progenitors, but not in multipotent progenitors. We do not detect effects on hematopoietic stem cells, or in peripheral blood. Notably, we do not detect severe myelodysplastic phenotypes or leukemia in this loss-of-function model. We conclude that Asxl1 is needed for normal hematopoiesis. The mild phenotypes observed may be because other Asxl genes have redundant function with Asxl1, or alternatively, MDS or oncogenic phenotypes may result from gain-of-function Asxl mutations caused by genomic amplification, gene fusion, or truncation of Asxl1.

The crystal structure of the ING5 PHD finger in complex with an H3K4me3 histone peptide

Chromatin dynamics regulates diverse nuclear processes and influences cellular viability and tumorigenesis. The discrete chromatin states are linked to covalent histone modifications that control the extent of DNA accessibility to transacting factors. One of the most common epigenetic modifications is methylation of histone H3 at lysine 4 (H3K4). Lys4 can be mono-, di-, or tri-methylated, and the tri-methylated mark (H3K4me3) is normally associated with euchromatin and active gene transcription.1,2 The inhibitor of growth (ING) family of tumor suppressors contain a C-terminal plant homeodomain (PHD) finger. This conserved zinc-binding module is found in many nuclear proteins including transcription factors, histone modifying enzymes, and ATP dependent chromatin remodeling complexes.3-5 A subset of PHD fingers has recently been shown to bind methylated and unmodified histone tails,5-9 with the H3K4me3 mark being specifically recognized by ING proteins. Unlike ING1 and ING2, which have been identified as components of histone deacetylase (HDAC) complexes, ING5 associates with histone acetyltransferase (HAT) complexes containing MOZ (monocytic leukemia zinc finger protein)/MORF (MOZ-related factor) and HBO1.10 To establish the structural basis of chromatin targeting by the HAT associated ING proteins, we determined the crystal structure of the ING5 PHD finger in complex with its histone target (H3K4me3). We also measured binding affinities for unmodified, mono-, di-, and tri-methylated histone peptides, and showed that both full-length ING5 and methylated H3K4 are essential for the acetyltransferase activity of the MOZ/MORF and HBO1 complexes. This functional data are the first direct evidence supporting the critical role of ING5 in directing the MOZ/MORF and HBO1 complexes to chromatin, which consequently increases the local HAT activity and stimulates chromatin remodeling.

Histone H3K4me3 Binding Is Required for the DNA Repair and Apoptotic Activities of ING1 Tumor Suppressor

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 Å-resolution crystal structure of the PHD–H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation–π contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignances, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

PHF23: A Novel Erythropoietin-Induced Gene Associated with AML and MDS.

Chromosomal translocations in a number of hematopoietic malignancies, including AML and MDS, result in the formation of NUP98 gene chimeras. Recently, fusion of NUP98 with a novel gene, PHF23 or plant homeodomain (PHD) finger 23, was reported in a cryptic translocation in a patient with AML (Reader et al., 2007). PHF23 is one of three known NUP98 fusion partners, associated with AML, which contain PHD finger motifs. In this study, we report a more detailed characterization of PHF23, which we originally identified by differential display PCR as an erythropoietin-induced gene in a murine model of terminal erythroid differentiation. In silico sequence analysis revealed a five exon gene containing a nuclear localization signal (NLS) as well as the C-terminal PHD motif. PHF23 mapped to chromosome 17p13.1, <450 kb from the TP53 gene, in a region frequently disrupted in cancer. Quantitative PCR analysis indicated Phf23 expression is not restricted to hematopoietic cells but demonstrates widespread tissue distribution, with increased expression levels in more proliferative tissues (∼6 fold increase in gene expression in the testis over mRNA levels in the liver). A ubiquitously expressed Phf23 splice variant (with mRNA concentrations ∼30–45% lower than full-length Phf23 gene expression levels) was generated using an alternative 3′ splice site within exon four, which exactly corresponded to the chr17 translocation breakpoint in the AML patient described. PHF23 is evolutionarily conserved, particularly within the PHD finger domain (>76% sequence identity between human and zebrafish PHD motifs), and is significantly related to another potentially oncogenic protein, PHF13/SPOC-1, associated with ovarian cancer. PHD motifs act as zinc fingers and the expanding family of PHD-containing proteins are implicated in chromatin regulation. Cellular localization studies using fluorescently tagged PHF23 indicated that both the full-length and spliced isoforms are located exclusively within the nucleus. We predicted that PHF23 has a similar structure to PHD motifs of known histone effector proteins, which influence transcription through recognition of specific epigenetic marks. This was demonstrated by immunoprecipitation and histone peptide binding assays showing that PHF23 associated with histone H3, primarily di- and tri-methylated H3K4, which are characteristic marks of active transcription. Microarray analysis of PHF23 mRNA expression levels in AML and MDS patient samples indicated no defined association with AML, however a positive correlation was demonstrated with a subset of MDS patients exhibiting erythroid dysplasia. This study suggests that PHF23, a novel histone effector protein, may play a role in aberrant transcription during defective hematopoiesis.