SANT Domain Research Articles

The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma.

Despite recent advances in therapeutic treatments, multiple myeloma (MM) remains an incurable malignancy. Epigenetic factors contribute to the initiation, progression, relapse, and clonal heterogeneity in MM, but our knowledge on epigenetic mechanisms underlying MM development is far from complete. The SAGA complex serves as a coactivator in transcription and catalyzes acetylation and deubiquitylation. Analyses of data sets in the Cancer Dependency Map Project revealed that many SAGA components are selective dependencies in MM. To define SAGA-specific functions, we focused on ADA2B, the only subunit in the lysine acetyltransferase (KAT) module that specifically functions in SAGA. Integration of RNA sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), and cleavage under targets and release using nuclease assay (CUT&RUN) results identified pathways directly regulated by ADA2B including MTORC1 signaling and oncogenic programs driven by MYC, E2F, and MM-specific MAF. We discovered that ADA2B is recruited to MAF and MYC gene targets, and that MAF shares a majority of its targets with MYC in MM cells. Furthermore, we found that the SANT domain of ADA2B is required for interaction with both GCN5 and PCAF acetyltransferases, incorporation into SAGA, and ADA2B protein stability. Our findings uncover previously unknown SAGA KAT module-dependent mechanisms controlling MM cell growth, revealing a vulnerability that might be exploited for future development of MM therapy.

Effects of MhMYB1 and MhMYB2 transcription factors on the monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq.

Transcription factors MhMYB1 and MhMYB2 correlate with monoterpenoid biosynthesis pathway in l-menthol chemotype of Mentha haplocalyx Briq, which could affect the contents of ( -)-menthol and ( -)-menthone. Mentha haplocalyx Briq., a plant with traditional medicinal and edible uses, is renowned for its rich essential oil content. The distinct functional activities and aromatic flavors of mint essential oils arise from various chemotypes. While the biosynthetic pathways of the main monoterpenes in mint are well understood, the regulatory mechanisms governing different chemotypes remain inadequately explored. In this investigation, we identified and cloned two transcription factor genes from the M. haplocalyx MYB family, namely MhMYB1 (PP236792) and MhMYB2 (PP236793), previously identified by our research group. Bioinformatics analysis revealed that MhMYB1 possesses two conserved MYB domains, while MhMYB2 contains a conserved SANT domain. Yeast one-hybrid (Y1H) analysis results demonstrated that both MhMYB1 and MhMYB2 interacted with the promoter regions of MhMD and MhPR, critical enzymes in the monoterpenoid biosynthesis pathway of M. haplocalyx. Subsequent virus-induced gene silencing (VIGS) of MhMYB1 and MhMYB2 led to a significant reduction (P < 0.01) in the relative expression levels of MhMD and MhPR genes in the VIGS groups of M. haplocalyx. In addition, there was a noteworthy decrease (P < 0.05) in the contents of ( -)-menthol and ( -)-menthone in the essential oil of M. haplocalyx. These findings suggest that MhMYB1 and MhMYB2 transcription factors play a positive regulatory role in ( -)-menthol biosynthesis, consequently influencing the essential oil composition in the l-menthol chemotype of M. haplocalyx. This study serves as a pivotal foundation for unraveling the regulatory mechanisms governing monoterpenoid biosynthesis in different chemotypes of M. haplocalyx.

Trichothecium roseum epigenetic reader Snt2 in phenotype and pathogenicity regulation

As an epigenetic reader, Snt2 is involved in the regulation of phenotype and pathogenicity of some filamentous fungi. However, whether Snt2 is involved in the regulation of phenotype and pathogenicity of Trichothecium roseum has not been reported. In this study, snt2-deleted strain of T. roseum and its complementation were constructed based on bioinformatics analysis to investigate the effects of Trsnt2 on the phenotype and pathogenicity of T. roseum. The results showed that the Snt2 protein in T. roseum has a high homology to that of Fusarium albosuccineum. The protein contains one BAH domain, two PHD domains, one SANT domain and one ELM2 domain. Deletion of Trsnt2 inhibited colony growth, reduced aerial hyphae, decreased polar growth, and increased hyphal branching of T. roseum. In addition, the deletion of Trsnt2 downregulated the expression of key genes related to sporulation, including TrbrlA, TrabaA, and TrwetA, reducing sporulation but increasing spore germination. The Trsnt2 deletion strain was more sensitive to cell wall stress and oxidative stress, but not to osmotic stress. In addition, Trsnt2 deletion downregulated the expression levels of PacC, ammonia synthesis-related genes (TrGDH2, TrAMET, TrGLT1, and TrMEPB), and extracellular enzyme-related genes (TrpelB, TrplyB, Trpme1, TrpgaX, TreglA, Trbgl1, Trcbh1, and Trcut1) in T. roseum. Deletion of Trsnt2 also reduced the pathogenicity of T. roseum on apple fruit. In conclusion, deletion of Trsnt2 inhibited the growth and development of T. roseum, reduced sensitivity of the strain to osmotic stress, but increased to cell wall and oxidative stress, and reduced the pathogenicity of the strain on apple fruit.

Identification, characterization, and CADD analysis of Plasmodium DMAP1 reveals it as a potential molecular target for new anti-malarial discovery

Developing drug resistance in the malaria parasite is a reason for apprehension compelling the scientific community to focus on identifying new molecular targets that can be exploited for developing new anti-malarial compounds. Despite the availability of the Plasmodium genome, many protein-coding genes in Plasmodium are still not characterized or very less information is available about their functions. DMAP1 protein is known to be essential for growth and plays an important role in maintaining genomic integrity and transcriptional repression in vertebrate organisms. In this study, we have identified a homolog of DMAP1 in P. falciparum. Our sequence and structural analysis showed that although PfDMAP1 possesses a conserved SANT domain, parasite protein displays significant structural dissimilarities from human homolog at full-length protein level as well as within its SANT domain. PPIN analysis of PfDMAP1 revealed it to be vital for parasite and virtual High-throughput screening of various pharmacophore libraries using BIOVIA platform-identified compounds that pass ADMET profiling and showed specific binding with PfDMAP1. Based on MD simulations and protein–ligand interaction studies two best hits were identified that could be novel potent inhibitors of PfDMAP1 protein. Communicated by Ramaswamy H. Sarma

Comprehensive analysis of MYB gene family and their expression under various stress conditions in Lilium pumilum

The MYB family proteins play crucial roles in regulating the metabolism for plant growth and development as well as stress responses, thus, have great potential in breeding novel plant varieties with improved traits such as abiotic stress tolerance. Although the MYB genes have extensively studied in various plant species, they have not yet largely explored in Lilium species, which has high ornamental and medicinal values, limiting the practices for breeding abiotic stress tolerant Lilium species. For improving the abiotic stress tolerance of Lilium species, we identified 23 MYB genes in Lilium pumilum (salt-tolerant Lilium species) and performed comprehensive characterization of structure prediction, phylogenetic relationships, conserved motifs, and time- and tissue-specific expression patterns. Phylogenetic relationships of the LpMYB transcription factors showed their diverse roles related to various developmental processes and environmental interactions in L. pumilum. Structure prediction and conserved motif analysis revealed that LpMYB transcription factors have the conserved domain such as SANT domain in common. The LpMYB genes revealed explicit time- and tissue-specific induction pattern upon ABA, cold, PEG, and salt treatment. Particularly, LpMYB1 showed the strongest induction response upon salt in roots of L. pumilum. LpMYB1 overexpression tobacco lines further exhibited the improved salt tolerance phenotypes, such as higher levels of chlorophyll, proline, SOD, POD, and CAT enzymes while lower levels of MDA. Taken together, this study identified novel LpMYB gene family in L. pumilum and contributed to building the preliminary knowledge for further in-depth characterization of the LpMYB transcription factors as well as breeding stress-tolerant Lilium species.

A SAGA Complex-Specific Dependency in Multiple Myeloma Orchestrates Oncogenic Transcriptional Programs

Multiple myeloma (MM) is a malignancy of antibody-producing plasma cells that reside within the bone marrow. Epigenetic factors contribute to the initiation, progression, relapse and clonal heterogeneity in MM, but our knowledge of epigenetic regulators involved in MM development is far from complete. Results from the Cancer Dependency Map (DepMap) Project revealed that several components of the SAGA complex are particularly important for MM cell growth and survival. The SAGA complex serves as a coactivator in transcription and also catalyzes histone acetylation and deubiquitination via its lysine acetyltransferase (KAT) and deubiquitinase (DUB) modules, respectively. Our analysis showed that each SAGA complex component is overexpressed in at least 50% of MM patients. To investigate SAGA-specific functions, we centered on ADA2B, a subunit in the KAT module that specifically functions in the SAGA complex. We observed reduced cell viability upon ADA2B knockdown and CRISPR/Cas9-mediated knockout in MM cell lines. To identify the underlying transcriptional pathways that ADA2B regulate, we performed transcriptome sequencing using MM cell lines with and without ADA2B depletion. Our results showed several oncogenic pathways, including MYC, E2F and MM-specific MAF oncogenic programs, are tightly regulated by ADA2B. We also discovered that the protein but not transcript levels of MAF and the KATs in SAGA, GCN5 and PCAF, as well as the global level of histone H3 lysine 9 acetylation (H3K9ac) are all prominently decreased upon ADA2B depletion. To investigate whether the global reduction in H3K9ac would alter chromatin accessibility, we performed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and found decreased accessibility at many cancer marker and cancer-promoting genes. To decipher the protein domains that are essential for ADA2B function in MM, we generated and expressed mutated versions of ADA2B in MM cell lines. Excitingly, mutations of core hydrophobic amino acid residues in the SANT domain disrupt interactions with GCN5 and PCAF and negatively affect protein stability of ADA2B. Hence, drug inhibitors that target the SANT domain could achieve suppression of ADA2B and MM cell growth. Our findings uncover novel epigenetic and transcriptional mechanisms controlling MM development, and suggest potential for new epigenetic inhibitors targeting SAGA-specific ADA2B as future treatment modalities for MM.

Abstract P1116: Histone Deacetylase Complexes Regulate Direct Cardiac Conversion From Human Fibroblasts

Direct cardiac reprogramming is a promising approach to generate functional cardiomyocytes from non-myocytes for regenerative medicine, drug screening, and disease modeling. This method involves the transformation of specialized cells into functional cardiomyocytes using specific transcription factors or molecular signals. Epigenetic modulators, such as histone deacetylase (HDAC) complexes, may play a critical role in chromatin remodeling underlying cardiac reprogramming. To investigate the role of HDAC complexes in direct cardiac conversion, induced cardiomyocyte-like cells (iCMs) were derived from human fibroblasts through ectopic expression of reprogramming factors, and the changes of histone acetylation were evaluated by western blotting. The results showed decreased levels of H3K9ac, H3K14ac, and H3K27ac along with direct cardiac reprogramming, suggesting the significant chromatin remodeling and gene regulation. A loss-of-function screen of all the components associated with HDAC complex was performed to determine the responsible histone acetylation modifiers. The screen results showed that most of the HDAC components are necessary for direct cardiac reprogramming. Further investigation focused on a newly identified HDAC complex, mitotic deacetylase complex (MiDAC), consisting of mitotic deacetylase associated SANT domain protein MiDEAS (ELMSAN1), DNTTIP1, and HDAC1. Knocking down each of the MiDAC components led to similar reprogramming repression and cell death, indicating that MiDAC plays a critical role in cardiac reprogramming. In addition, the expression analysis of DNTTIP1 and HDAC1 showed a gradual decrease throughout the conversion, while ELMSAN1 diminished at the early stage of reprogramming, suggesting that ELMSAN1 is likely required for MiDAC stability. Furthermore, overexpression of ELMSAN1 significantly improved reprogramming efficiency and led to the increase of H3K9ac, H3K14ac, and H3K27ac levels globally, suggesting that ELMSAN1 and MiDAC might be responsible for the regulation of such histone modifications during the process. Overall, our findings provide novel insights into the epigenetic regulation of direct cardiac reprogramming and reveal the potential role of MiDAC in this process.

Cloning and functional analysis of RADIALIS-like 1 gene from Antirrhinum majus.

MYB is one of the largest transcription factor families in plants. Among them, the R3-MYB transcription factor RADIALIS (RAD) plays a very important role in the flowers development in Antirrhinum majus. In this study, a R3-MYB gene similar to RAD was found by analyzing the genome of A. majus, which was named AmRADIALIS-like 1 (AmRADL1). The gene function was predicted through bioinformatics. The relative expression levels in different tissues and organs of wild-type A. majus were analyzed by qRT-PCR. AmRADL1 was overexpressed in A. majus, and the transgenic plants were analyzed by morphological observation and histological staining. The results showed that the open reading frame (ORF) of AmRADL1 gene was 306 bp in length, encoding 101 amino acids. It has typical SANT domain, and the C-terminal contains a CREB motif, which was highly homologous to tomato SlFSM1. The results of qRT-PCR showed that AmRADL1 was expressed in roots, stems, leaves and flowers, and the expression level was higher in flowers. Further analysis of its expression in different floral organs showed that AmRADL1 had the highest expression in carpel. The results of histological staining analysis of the transgenic plants showed that compared with the wild type, although the size of the carpel cells of the transgenic plants did not change significantly, the placenta area in the carpel became smaller and the number of cell decreased. In summary, AmRADL1 may be involved in the regulation of carpel development, but the specific mechanism of action in carpel remains to be further studied.

Overexpression of the cotton trihelix transcription factor GhGT23 in Arabidopsis mediates salt and drought stress tolerance by binding to GT and MYB promoter elements in stress-related genes.

Cotton (Gossypium hirsutum L.) is the world's most economically valuable textile crop. However, cotton plants are often subjected to numerous abiotic stresses that can dramatically limit yield. Trihelix transcription factors (TTFs) play important roles in abiotic stress responses in many plant species, and efforts to better understand their roles in cotton abiotic stress responses are ongoing. In this study, a member of the cotton TTF family (GhGT23) was functionally characterized. This protein contains a SANT domain and is a member of the SIP subfamily of TTF proteins. GhGT23 was significantly (p < 0.05) and highly expressed in cotton fiber compared to relatively low expression in other tissues. A significant (p < 0.05) increase in GhGT23 expression occurred in cotton seedlings within 12 hours of drought, salt, and ABA exposure. The GhGT23 protein localized in the nucleus but exhibited no signs of transactivation activity. GhGT23 overexpression in Arabidopsis conferred enhanced drought and salt stress tolerance. The expression of stress-related genes was higher in transgenic Arabidopsis expressing GhGT23 than in wild-type plants subjected to salt stress. The results of electrophoretic mobility shift assay revealed that GhGT23 could bind to the GT cis-elements GT-1Box (Box II), GT2-Box, GT3-Box, GT-3a (Site1-type), GT-3b, and Box as well as the MYB cis-elements MBS1 and MRE4. Our results demonstrate that GhGT23 positively regulates salt and drought stress responses, possibly by enhancing the expression of stress-related genes.

Trihelix Transcriptional Factor GhGT26 of Cotton Enhances Salinity Tolerance in Arabidopsis.

Cotton (Gossypium hirsutum L.), the most important textile crop worldwide, often encounters abiotic stress during its growing season and its productivity is significantly limited by adverse factors. Trihelix transcription factors (also known as GT factors) are important proteins involved in the morphological development and responses to abiotic stress in plants. However, their functions and molecular mechanisms in the cotton toward abiotic stress response remain unclear. In this study, a member (GhGT26) of the cotton Trihelix family was functionally characterized in the model plant Arabidopsis. This protein containing a SANT domain belongs to the GT-1 subgroup of trihelix proteins. GhGT26 was widely expressed in tissues (with the highest level in flower) and responded to high salt and ABA treatments at the transcriptional level. Using the Arabidopsis protoplast assay system, we found that the GhGT26 protein was located in the cell nuclei. The EMSA assay revealed that the GhGT26 protein could bind to the Site1-type GT cis elements (GT-3a) and MYB elements MRE3 and MRE4. The overexpression of GhGT26 improved plant tolerance to salt stress in transgenic Arabidopsis plants. Although ABA inhibits root elongation, the statistical analysis revealed that the root lengths of GhGT26-overexpressing Arabidopsis were the same as the wild plants after ABA treatment. Our results demonstrate that GhGT26 positively regulates salt stress via ABA-independent pathways. This evidence suggests that the GhGT26 may participate in the regulation of stress tolerance in cotton.

Transcriptome Analysis of MYB Genes and Patterns of Anthocyanin Accumulation During Seed Development in Wheat.

Plants accumulate key metabolites as a response of biotic/abiotic stress conditions. In seed coats, anthocyanins, carotenoids, and chlorophylls can be found. They have been associated as important antioxidants that affect germination. In wheat, anthocyanins can impart the seed coat color which have been recognized as health-promoting nutrients. Transcription factors act as master regulators of cellular processes. Transcription complexes such as MYB-bHLH-WD40 (MBW) regulate the expression of multiple target genes in various plant species. In this study, the spatiotemporal accumulation of seed coat pigments in different developmental stages (10, 20, 30, and 40 days after pollination) was analyzed using cryo-cuts. Moreover, the accumulation of phenolic, anthocyanin, and chlorophyll contents was quantified, and the expression of flavonoid biosynthetic genes was evaluated. Finally, transcriptome analysis was performed to analyze putative MYB genes related to seed coat color, followed by further characterization of putative genes. TaTCL2, an MYB gene, was cloned and sequenced. It was determined that TaTCL2 contains a SANT domain, which is often present in proteins participating in the response to anthocyanin accumulation. Moreover, TaTCL2 transcript levels were shown to be influenced by anthocyanin accumulation during grain development. Interaction network analysis showed interactions with GL2 (HD-ZIP IV), EGL3 (bHLH), and TTG1 (WD40). The findings of this study elucidate the mechanisms underlying color formation in Triticum aestivum L. seed coats.

Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis

Owing to the lack of genetic animal models that adequately recreate key clinical characteristics of cirrhosis, the molecular pathogenesis of cirrhosis has been poorly characterized, and treatments remain limited. Hence, we aimed to better elucidate the pathological mechanisms of cirrhosis using a novel murine model. We report on the first murine genetic model mimicking human cirrhosis induced by hepatocyte-specific elimination of microspherule protein 1 (MCRS1), a member of non-specific lethal (NSL) and INO80 chromatin-modifier complexes. Using this genetic tool with other mouse models, cell culture and human samples, combined with quantitative proteomics, single nuclei/cell RNA sequencing and chromatin immunoprecipitation assays, we investigated mechanisms of cirrhosis. MCRS1 loss in mouse hepatocytes modulates the expression of bile acid (BA) transporters - with a pronounced downregulation of Na+-taurocholate cotransporting polypeptide (NTCP) - concentrating BAs in sinusoids and thereby activating hepatic stellate cells (HSCs) via the farnesoid X receptor (FXR), which is predominantly expressed in human and mouse HSCs. Consistently, re-expression of NTCP in mice reduces cirrhosis, and genetic ablation of FXR in HSCs suppresses fibrotic marks in mice and invitro cell culture. Mechanistically, deletion of a putative SANT domain from MCRS1 evicts histone deacetylase 1 from its histone H3 anchoring sites, increasing histone acetylation of BA transporter genes, modulating their expression and perturbing BA flow. Accordingly, human cirrhosis displays decreased nuclear MCRS1 and NTCP expression. Our data reveal a previously unrecognized function of MCRS1 as a critical histone acetylation regulator, maintaining gene expression and liver homeostasis. MCRS1 loss induces acetylation of BA transporter genes, perturbation of BA flow, and consequently, FXR activation in HSCs. This axis represents a central and universal signaling event in cirrhosis, which has significant implications for cirrhosis treatment. By genetic ablation of MCRS1 in mouse hepatocytes, we generate the first genetic mouse model of cirrhosis that recapitulates human features. Herein, we demonstrate that the activation of the bile acid/FXR axis in liver fibroblasts is key in cirrhosis development.

Abstract PO-048: A novel chromatin remodeling domain of keratin 17 regulates transcription and promotes tumor aggression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by two molecular subtypes, of which the basal-like subtype is associated with the worst survival and is highly resistant to first-line chemotherapy. We previously reported that keratin 17 (K17), a component of the molecular signature of the basal-like subtype, is a negative prognostic and predictive biomarker, whose overexpression results in chemoresistance and shortened patient survival. Here, we aimed to uncover the mechanistic role of K17 in driving cancer aggression and explore the therapeutic opportunities in K17-expressing PDAC. Beyond its role as a cytoskeletal protein, K17 can solubilize from the filamentous form and translocate into the nucleus, where it impacts multiple cellular properties. Previous reports suggest that nuclear K17 regulates cell-cycle progression and gene expression. However, the detailed mechanisms through which K17 modulates gene expression remains unexplored. Through domain-prediction analyses, we identified a novel chromatin-remodeling (SANT) domain in K17 that may interact with histones to regulate chromatin structure. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) revealed that K17 positive cells had comparably accessible regions relative to K17 negative cells. However, the deletion of the K17 SANT domain (dSANT) decreased chromatin accessibility, suggesting that nuclear K17 impacts chromatin structure via the SANT domain. With RNA sequencing (RNA-seq) pathway enrichment analysis, we found that K17 positive cells had differential expressed genes that are associated with cell signaling, immune regulation, and metabolic pathways. More specifically, by comparing wild type K17 and dSANT K17-expressing cells, we found that the SANT domain impacts a wide variety of immune regulatory pathways, many of which affect the tumor microenvironment. We are now performing chromatin immunoprecipitation sequencing (ChIP-seq) with acetylation of lysine 27 on histone H3 protein subunit (H3K27ac), to reveal differential bindings that are impacted by K17 and the SANT domain. Importantly, mice bearing orthotopic xenografts of PDACs with K17-SANT deletion showed survival comparable to those with K17 negative tumors and had significantly longer survival than those with wild-type K17. In summary, we identified a novel chromatin remodeling domain in K17 that could be explored as a target for the development of a biomarker-based treatment for PDAC. Citation Format: Chun-Hao Pan, Robert Tseng, Simon J. Hogg, Gabriella Baraks, Cindy V. Leiton, Lucia Roa-Peña, Natalia Marchenko, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. A novel chromatin remodeling domain of keratin 17 regulates transcription and promotes tumor aggression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-048.

Genetic polymorphism in the POU1F1 gene in Kalahari Red and two Nigerian goat breeds and their relationship with litter size

POU1F1 gene controls cell differentiation and animal growth by binding to target DNA promoter sequence, thereby auto-regulating its own expression and expression of growth hormone (GH), prolactin (PRL) and thyroid-stimulating hormone beta sub-unit (TSHβ) genes. Therefore, the exploration of caprine POU1F1 gene polymorphisms may be vital in the formulation of conservation and breed improvement strategies. In this study, POU1F1 gene was characterized for sequence polymorphisms in 366 individuals from two Nigerian goat breeds ((West African Dwarf (WAD) and Red Sokoto (RS)) and one South African goat breed (Kalahari (KR)). The effects of polymorphisms on litter size were investigated using linear mixed model. Two intronic mutations (g.306G&gt;A and g.11236C&gt;T) were identified. However, no significant association was found between the Single Nucleotide Polymorphisms (SNPs) and litter size in the three populations. The genetic distance based on POU1F1 investigated region revealed that the two Nigerian breeds and the South African breed were identical (pairwise genetic distance of 0.00). Phylogenetic tree constructed from the pairwise distance clustered the three breeds into a single clade with the two Nigerian goat breeds having a more recent common ancestor. Structural analysis of the POU1F1 protein confirmed that Pit-Oct-Unc transcription factors domain (POU) and Homeodomain (HOX) domains are conserved in mammals, with several overlapping sub-domains across the same region in all the three populations. We found a subdomain Subfamily of SANT domain or myb/SANT-like domain in Adf-1 (MADF) in goat, cattle, buffalo and camel that has not been reported in mammals

The uncharacterized SANT and BTB domain-containing protein SANBR inhibits class switch recombination

Class switch recombination (CSR) is the process by which B cells switch production from IgM/IgD to other immunoglobulin isotypes, enabling them to mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line, we have identified a novel, uncharacterized protein of 82kD (KIAA1841, NM_027860), which we have named SANBR (SANT and BTB domain regulator of CSR), as a negative regulator of CSR. The purified, recombinant BTB domain of SANBR exhibited characteristic properties such as homodimerization and interaction with corepressor proteins, including HDAC and SMRT. Overexpression of SANBR inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Future investigations to identify transcriptional targets of SANBR in B cells will reveal further insights into the specific mechanisms by which SANBR regulates CSR as well as fundamental gene regulatory activities of this protein.

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT and YARP–NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single -helix that makes multiple contacts with -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.

Rap1-mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence.

Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for new Rap1 targets at senescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392–394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1SHY), and thus disrupts Rap1‐H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1‐H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1‐mediated nucleosome displacement and gene activation. Remarkably, RAP1SHY does not affect the pace of senescence‐related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling

Nucleosomes are the fundamental building blocks ofchromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.

Structure and evolution of the 4-helix bundle domain of Zuotin, a J-domain protein co-chaperone of Hsp70.

The J-domain protein Zuotin is a multi-domain eukaryotic Hsp70 co-chaperone. Though it is primarily ribosome-associated, positioned at the exit of the 60S subunit tunnel where it promotes folding of nascent polypeptide chains, Zuotin also has off-ribosome functions. Domains of Zuotin needed for 60S association and interaction with Hsp70 are conserved in eukaryotes. However, whether the 4-helix bundle (4HB) domain is conserved remains an open question. We undertook evolutionary and structural approaches to clarify this issue. We found that the 4HB segment of human Zuotin also forms a bundle of 4 helices. The positive charge of Helix I, which in Saccharomyces cerevisiae is responsible for interaction with the 40S subunit, is particularly conserved. However, the C-termini of fungal and human 4HBs are not similar. In fungi the C-terminal segment forms a plug that folds back into the bundle; in S. cerevisiae it plays an important role in bundle stability and, off the ribosome, in transcriptional activation. In human, C-terminal helix IV of the 4HB is extended, protruding from the bundle. This extension serves as a linker to the regulatory SANT domains, which are present in animals, plants and protists, but not fungi. Further analysis of Zuotin sequences revealed that the plug likely arose as a result of genomic rearrangement upon SANT domain loss early in the fungal lineage. In the lineage leading to S. cerevisiae, the 4HB was subjected to positive selection with the plug becoming increasingly hydrophobic. Eventually, these hydrophobic plug residues were coopted for a novel regulatory function—activation of a recently emerged transcription factor, Pdr1. Our data suggests that Zuotin evolved off-ribosome functions twice—once involving SANT domains, then later in fungi, after SANT domain loss, by coopting the hydrophobic plug. Zuotin serves as an example of complex intertwining of molecular chaperone function and cell regulation.

TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major.

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.