RNA Polymerase II-dependent Transcription Research Articles

Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders.

The TATA-box binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in males cause X-linked intellectual disability, a neurodevelopmental disorder, and TAF1 is dysregulated in X-linked Dystonia-Parkinsonism, a neurodegenerative disorder. However, this field has suffered from the lack of a genetic mouse model of TAF1 disease to explore mammalian mechanism and treatments. Here, we generated and validated a conditional cre-lox allele, and the first ubiquitous Taf1 knock-out mouse. We discovered that Taf1 deletion in males was embryonically lethal, which may explain why no human null-variants have been identified. In the brains of Taf1 heterozygous females, no differences were found in gross structure, overall expression, and protein localization, suggesting extreme skewed X-inactivation towards the non-mutant chromosome. Nevertheless, these female mice exhibited a significant increase in weight, weight with age, and reduced movement, suggesting a small subset of neurons has been negatively impacted by Taf1 loss. Finally, this new mouse may be a future platform for the development of TAF1 disease therapeutics.

SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription.

Dynamic regulation of phosphorylation and dephosphorylation of histones is essential for eukaryotic transcription, but the enzymes engaged in histone dephosphorylation are not fully explored. Here, we show that the tyrosine phosphatase SHP-1 dephosphorylates histone H2B and plays a critical role during transition from the initiation to the elongation stage of transcription. Nuclear-localized SHP-1 is associated with the Paf1 complex at chromatin and dephosphorylates H2B at tyrosine 121. Moreover, knockout of SHP-1, or expression of a mutant mimicking constitutive phosphorylation of H2B Y121, leads to a reduction in genome-wide H2B ubiquitination, which subsequently causes defects in RNA polymerase II-dependent transcription. Mechanistically, we demonstrate that Y121 phosphorylation precludes H2B's interaction with the E2 enzyme, indicating that SHP-1-mediated dephosphorylation of this residue may be a prerequisite for efficient H2B ubiquitination. Functionally, we find that SHP-1-mediated H2B dephosphorylation contributes to maintaining basal autophagic flux in cells through the efficient transcription of autophagy and lysosomal genes. Collectively, our study reveals an important modification of histone H2B regulated by SHP-1 that has a role during eukaryotic transcription.

Abstract 1115: BET bromodomain inhibition synergizes with MEK inhibitors in uveal melanoma

Abstract Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Approximately 50% of the patients develop hepatic metastasis and the median survival rate is 12 months. Currently available drugs have shown limited clinical activity in patients with UM and there is an urgent need for new effective therapies. UM is characterized by activating mutations of G protein q/11, mutations in BAP1, and amplification of the oncogene MYC. Epigenetic dysregulation plays a critical role in UM pathogenesis, and we have demonstrated that Gnaq/11 mutant UM cells are addicted to BRD4 activity. BRD4 is a key regulator of transcriptional elongation by recruiting positive transcriptional elongation factor complex (PTEFb) to chromatin and activating RNA polymerase II-dependent transcription. While BET inhibitors have shown potent activity in pre-clinical studies, single-agent BET inhibitors exhibited limited efficacy in advanced UM clinical trials. MAPK signaling in constitutively active in Gnaq/11 mutant UM cells and previous studies have shown that the MAPK pathway mediates resistance to BET inhibition. Therefore, we sought to combine MEK and BET inhibitors in a panel of UM cells. We found synergistic activities with a variety of structurally distinct MEK inhibitors including trametinib and binimetinib in combination with BET inhibitors, such as I-BET and PLX2853. These combinations induced a greater reduction in cell viability and significant apoptosis compared to either monotherapy, in both parental and BET inhibitor-resistant cells. Western blot studies were performed to evaluate the mechanism of the combination treatment with BET and MEK inhibition and demonstrated a marked downregulation of p-ERK, MYC, Bcl-xL and upregulation of BIM with the combination therapy. Similarly, the combination treatment resulted in greater inhibition of tumor growth compared to either BET or MEK inhibition alone in a Gnaq mutant xenograft model. In conclusion, our studies suggest that co-targeting of MEK and BET proteins may be required to maximize the responses of UM cells to BET inhibitors. Clinical trials with the combination of BET and MEK inhibitors are warranted. Citation Format: Grazia Ambrosini, Elgilda Musi, Gary K. Schwartz. BET bromodomain inhibition synergizes with MEK inhibitors in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1115.

BRLF1-dependent viral and cellular transcriptomes and transcriptional regulation during EBV primary infection in B lymphoma cells

The immediate-early protein BRLF1 plays important roles in lytic infection of Epstein-Barr virus (EBV), in which it activates lytic viral transcription and replication. However, knowledge of the influence of BRLF1 on cellular gene expression and transcriptional reprogramming during the early lytic cycle remains limited. In the present study, deep RNA-sequencing analysis identified all differentially expressed genes (DEGs) and alternative splicing in B lymphoma cells subjected to wild-type and BRLF1-deficient EBV primary infection. The BRLF1-dependent cellular DEGs were annotated, and major differentially enriched pathways were related to DNA replication and transcription, immune and inflammatory responses, cytokine-receptor interactions and chemokine signaling and metabolic processes. Furthermore, analysis of BRLF1-binding proteins by mass spectrometry shows that BRLF1 binds to and cooperates with several transcription factors and components of the spliceosome and then influences both RNA polymerase II-dependent transcription and pre-mRNA splicing. The RTA-binding RRE motifs or specific motifs of unique cooperative transcription factors in viral and cellular DEG promoter regions indicate that BRLF1 employs different strategies for regulating viral and cellular transcription. Thus, our study characterized BRLF1-dependent cellular and viral transcriptional profile during primary infection and then revealed the comprehensive virus-cell interaction and alterations of transcription during EBV primary infection and lytic replication.

Dynein and muskelin control myosin VI delivery towards the neuronal nucleus

SummaryProtein transport toward the nucleus is important for translating molecular signals into gene expression changes. Interestingly, the unconventional motor protein myosin VI regulates RNA polymerase II-dependent gene transcription. Whether actin-filament-dependent myosins are actively transported to nuclear compartments remains unknown.Here, we report that neurons also contain myosin VI inside their nucleus. Notably, nuclear appearance of this actin-dependent motor depends on functional cytoplasmic dynein, a minus end-directed microtubule motor. We find that the trafficking factor muskelin assists in the formation of dynein-myosin VI interactions and further localizes to nuclear foci, enriched in the myosin. Impairment of dynein, but not myosin VI function, reduces nuclear muskelin levels. In turn, muskelin represents a critical determinant in regulating myosin VI nuclear targeting.Our data reveal that minus end-directed microtubule transport determines myosin VI subcellular localization. They suggest a pathway of cytoplasm-to-nucleus trafficking that requires muskelin and is based on dynein-myosin cross talk.

A Mini Review on Gene Expression

A DNA molecule is more than just a long, monotonous string of nucleotides. Instead, it is divided into functional units known as genes. All cells use information encoded in their DNA to control or regulate protein synthesis. Gene refers to the process of turning on a to produce RNA and protein. All known life— eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), and viruses—use to generate the macromolecular machinery for life. Gene is the most fundamental level at which the genotype gives rise to t in genetics. The regulation of is a highly complex process. Strictly speaking, gene expression refers to the process of a being activated until a mature protein is found in its corresponding compartment to perform its function and contribute to the of a cell's phenotype. The purpose of the studies is to detect and quantify messenger RNA (mRNA) levels of a specific gene. The rapid activation of in response to stimuli is largely controlled by RNA polymerase II-dependent transcription. Gene microarrays are being used to study RNA and may be used to derive profiles/signatures associated with tumour and normal tissue radio response. The events that occur during the transcription cycle in eukaryotes that are important for the rapid and specific activation of in response to external stimuli are discussed in this review.

Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations.

Although originally thought to be silent chromosomal regions, centromeres are instead actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA-smFISH foci levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere-nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B-H4K8ac-BRD4 axis.

Centromeres are genomic regions essential for faithful chromosome segregation. Transcription of noncoding RNA (ncRNA) at centromeres is important for their formation and functions. Here, we report the molecular mechanism by which the transcriptional regulator ZFAT controls the centromeric ncRNA transcription in human and mouse cells. Chromatin immunoprecipitation with high-throughput sequencing analysis shows that ZFAT binds to centromere regions at every chromosome. We find a specific 8-bp DNA sequence for the ZFAT-binding motif that is highly conserved and widely distributed at whole centromere regions of every chromosome. Overexpression of ZFAT increases the centromeric ncRNA levels at specific chromosomes, whereas its silencing reduces them, indicating crucial roles of ZFAT in centromeric transcription. Overexpression of ZFAT increases the centromeric levels of both the histone acetyltransferase KAT2B and the acetylation at the lysine 8 in histone H4 (H4K8ac). siRNA-mediated knockdown of KAT2B inhibits the overexpressed ZFAT-induced increase in centromeric H4K8ac levels, suggesting that ZFAT recruits KAT2B to centromeres to induce H4K8ac. Furthermore, overexpressed ZFAT recruits the bromodomain-containing protein BRD4 to centromeres through KAT2B-mediated H4K8ac, leading to RNA polymerase II-dependent ncRNA transcription. Thus, ZFAT binds to centromeres to control ncRNA transcription through the KAT2B–H4K8ac–BRD4 axis.

Prefoldin-like Bud27 influences the transcription of ribosomal components and ribosome biogenesis in Saccharomyces cerevisiae.

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway.

Long noncoding RNAs in cancer: From discovery to therapeutic targets.

Long noncoding RNAs (lncRNAs) have recently gained considerable attention as key players in biological regulation; however, the mechanisms by which lncRNAs govern various disease processes remain mysterious and are just beginning to be understood. The ease of next-generation sequencing technologies has led to an explosion of genomic information, especially for the lncRNA class of noncoding RNAs. LncRNAs exhibit the characteristics of mRNAs, such as polyadenylation, 5' methyl capping, RNA polymerase II-dependent transcription, and splicing. These transcripts comprise more than 200 nucleotides (nt) and are not translated into proteins. Directed interrogation of annotated lncRNAs from RNA-Seq datasets has revealed dramatic differences in their expression, largely driven by alterations in transcription, the cell cycle, and RNA metabolism. The fact that lncRNAs are expressed cell- and tissue-specifically makes them excellent biomarkers for ongoing biological events. Notably, lncRNAs are differentially expressed in several cancers and show a distinct association with clinical outcomes. Novel methods and strategies are being developed to study lncRNA function and will provide researchers with the tools and opportunities to develop lncRNA-based therapeutics for cancer.

Epigenetics and transcription regulation during eukaryotic diversification: the saga of TFIID.

The basal transcription factor TFIID is central for RNA polymerase II-dependent transcription. Human TFIID is endowed with chromatin reader and DNA-binding domains and protein interaction surfaces. Fourteen TFIID TATA-binding protein (TBP)-associated factor (TAF) subunits assemble into the holocomplex, which shares subunits with the Spt-Ada-Gcn5-acetyltransferase (SAGA) coactivator. Here, we discuss the structural and functional evolution of TFIID and its divergence from SAGA. Our orthologous tree and domain analyses reveal dynamic gains and losses of epigenetic readers, plant-specific functions of TAF1 and TAF4, the HEAT2-like repeat in TAF2, and, importantly, the pre-LECA origin of TFIID and SAGA. TFIID evolution exemplifies the dynamic plasticity in transcription complexes in the eukaryotic lineage.

COMPASS Ascending: Emerging clues regarding the roles of MLL3/KMT2C and MLL2/KMT2D proteins in cancer.

The KMT2 (lysine methyltransferase) family of histone modifying proteins play essential roles in regulating developmental pathways, and mutations in the genes encoding these proteins have been strongly linked to many blood and solid tumor cancers. The KMT2A-D proteins are histone 3 lysine 4 (H3K4) methyltransferases embedded in large COMPASS-like complexes important for RNA Polymerase II-dependent transcription. KMT2 mutations were initially associated with pediatric Mixed Lineage Leukemias (MLL) and found to be the result of rearrangements of the MLL1/KMT2A gene at 11q23. Over the past several years, large-scale tumor DNA sequencing studies have revealed the potential involvement of other KMT2 family genes, including heterozygous somatic mutations in the paralogous MLL3/KMT2C and MLL2(4)/KMT2D genes that are now among the most frequently associated with human cancer. Recent studies have provided a better understanding of the potential roles of disrupted KMT2C and KMT2D family proteins in cell growth aberrancy. These findings, together with an examination of cancer genomics databases provide new insights into the contribution of KMT2C/D proteins in epigenetic gene regulation and links to carcinogenesis.

Maternal Nanos inhibits Importin-α2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline.

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. Here, we show that Nos represses somatic gene expression by inhibiting translation of maternal importin-α2 (impα2) mRNA. Mis-expression of Impα2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, we propose that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a ‘double-lock’ mechanism to inhibit somatic gene expression in germline progenitors.

RNA Polymerase II-Dependent Transcription Initiated by Selectivity Factor 1: A Central Mechanism Used by MLL Fusion Proteins in Leukemic Transformation.

Cancer cells transcribe RNAs in a characteristic manner in order to maintain their oncogenic potentials. In eukaryotes, RNA is polymerized by three distinct RNA polymerases, RNA polymerase I, II, and III (RNAP1, RNAP2, and RNAP3, respectively). The transcriptional machinery that initiates each transcription reaction has been purified and characterized. Selectivity factor 1 (SL1) is the complex responsible for RNAP1 pre-initiation complex formation. However, whether it plays any role in RNAP2-dependent transcription remains unclear. Our group previously found that SL1 specifically associates with AF4 family proteins. AF4 family proteins form the AEP complex with ENL family proteins and the P-TEFb elongation factor. Similar complexes have been independently characterized by several different laboratories and are often referred to as super elongation complex. The involvement of AEP in RNAP2-dependent transcription indicates that SL1 must play an important role in RNAP2-dependent transcription. To date, this role of SL1 has not been appreciated. In leukemia, AF4 and ENL family genes are frequently rearranged to form chimeric fusion genes with MLL. The resultant MLL fusion genes produce chimeric MLL fusion proteins comprising MLL and AEP components. The MLL portion functions as a targeting module, which specifically binds chromatin containing di-/tri-methylated histone H3 lysine 36 and non-methylated CpGs. This type of chromatin is enriched at the promoters of transcriptionally active genes which allows MLL fusion proteins to selectively bind to transcriptionally-active/CpG-rich gene promoters. The fusion partner portion, which recruits other AEP components and SL1, is responsible for activation of RNAP2-dependent transcription. Consequently, MLL fusion proteins constitutively activate the transcription of previously-transcribed MLL target genes. Structure/function analysis has shown that the ability of MLL fusion proteins to transform hematopoietic progenitors depends on the recruitment of AEP and SL1. Thus, the AEP/SL1-mediated gene activation pathway appears to be the central mechanism of MLL fusion-mediated transcriptional activation. However, the molecular mechanism by which SL1 activates RNAP2-dependent transcription remains largely unclear. This review aims to cover recent discoveries of the mechanism of transcriptional activation by MLL fusion proteins and to introduce novel roles of SL1 in RNAP2-dependent transcription by discussing how the RNAP1 machinery may be involved in RNAP2-dependent gene regulation.

Cyclin C: The Story of a Non-Cycling Cyclin.

The class I cyclin family is a well-studied group of structurally conserved proteins that interact with their associated cyclin-dependent kinases (Cdks) to regulate different stages of cell cycle progression depending on their oscillating expression levels. However, the role of class II cyclins, which primarily act as transcription factors and whose expression remains constant throughout the cell cycle, is less well understood. As a classic example of a transcriptional cyclin, cyclin C forms a regulatory sub-complex with its partner kinase Cdk8 and two accessory subunits Med12 and Med13 called the Cdk8-dependent kinase module (CKM). The CKM reversibly associates with the multi-subunit transcriptional coactivator complex, the Mediator, to modulate RNA polymerase II-dependent transcription. Apart from its transcriptional regulatory function, recent research has revealed a novel signaling role for cyclin C at the mitochondria. Upon oxidative stress, cyclin C leaves the nucleus and directly activates the guanosine 5’-triphosphatase (GTPase) Drp1, or Dnm1 in yeast, to induce mitochondrial fragmentation. Importantly, cyclin C-induced mitochondrial fission was found to increase sensitivity of both mammalian and yeast cells to apoptosis. Here, we review and discuss the biology of cyclin C, focusing mainly on its transcriptional and non-transcriptional roles in tumor promotion or suppression.

Transcriptional targeting of oncogene addiction in medullary thyroid cancer.

Metastatic medullary thyroid cancer (MTC) is incurable and FDA-approved kinase inhibitors that include oncogenic RET as a target do not result in complete responses. Association studies of human MTCs and murine models suggest that the CDK/RB pathway may be an alternative target. The objective of this study was to determine if CDKs represent therapeutic targets for MTC and to define mechanisms of activity. Using human MTC cells that are either sensitive or resistant to vandetanib, we demonstrate that palbociclib (CDK4/6 inhibitor) is not cytotoxic to MTC cells but that they are highly sensitive to dinaciclib (CDK1/2/5/9 inhibitor) accompanied by reduced CDK9 and RET protein and mRNA levels. CDK9 protein was highly expressed in 83 of 83 human MTCs and array-comparative genomic hybridization had copy number gain in 11 of 30 tumors. RNA sequencing demonstrated that RNA polymerase II-dependent transcription was markedly reduced by dinaciclib. The CDK7 inhibitor THZ1 also demonstrated high potency and reduced RET and CDK9 levels. ChIP-sequencing using H3K27Ac antibody identified a superenhancer in intron 1 of RET. Finally, combined inhibition of dinaciclib with a RET kinase inhibitor was synergistic. In summary, we have identified what we believe is a novel mechanism of RET transcription regulation that potentially can be exploited to improve RET therapeutic targeting.

Disease-relevant signalling-pathways in head and neck cancer: Taspase1\u2019s proteolytic activity fine-tunes TFIIA function

Head and neck cancer (HNC) is the seventh most common malignancy in the world and its prevailing form, the head and neck squamous cell carcinoma (HNSCC), is characterized as aggressive and invasive cancer type. The transcription factor II A (TFIIA), initially described as general regulator of RNA polymerase II-dependent transcription, is part of complex transcriptional networks also controlling mammalian head morphogenesis. Posttranslational cleavage of the TFIIA precursor by the oncologically relevant protease Taspase1 is crucial in this process. In contrast, the relevance of Taspase1-mediated TFIIA cleavage during oncogenesis of HNSCC is not characterized yet. Here, we performed genome-wide expression profiling of HNSCC which revealed significant downregulation of the TFIIA downstream target CDKN2A. To identify potential regulatory mechanisms of TFIIA on cellular level, we characterized nuclear-cytoplasmic transport and Taspase1-mediated cleavage of TFIIA variants. Unexpectedly, we identified an evolutionary conserved nuclear export signal (NES) counteracting nuclear localization and thus, transcriptional activity of TFIIA. Notably, proteolytic processing of TFIIA by Taspase1 was found to mask the NES, thereby promoting nuclear localization and transcriptional activation of TFIIA target genes, such as CDKN2A. Collectively, we here describe a hitherto unknown mechanism how cellular localization and Taspase1 cleavage fine-tunes transcriptional activity of TFIIA in HNSCC.

Nuclear fragments of the neural cell adhesion molecule NCAM with or without polysialic acid differentially regulate gene expression

The neural cell adhesion molecule (NCAM) is the major carrier of polysialic acid (PSA) which modulates NCAM functions of neural cells at the cell surface. In previous studies, we have shown that stimulation of cultured neurons with surrogate NCAM ligands leads to the generation and nuclear import of PSA-lacking and -carrying NCAM fragments. Here, we show that the nuclear import of the PSA-carrying NCAM fragment is mediated by positive cofactor 4 and cofilin, which we identified as novel PSA-binding proteins. In the nucleus, the PSA-carrying NCAM fragment interacts via PSA with PC4 and cofilin, which are involved in RNA polymerase II-dependent transcription. Microarray analysis revealed that the nuclear PSA-carrying and -lacking NCAM fragments affect expression of different genes. By qPCR and immunoblot analysis we verified that the nuclear PSA-carrying NCAM fragment increases mRNA and protein expression of nuclear receptor subfamily 2 group F member 6, whereas the PSA-lacking NCAM fragment increases mRNA and protein expression of low density lipoprotein receptor-related protein 2 and α-synuclein. Differential gene expression evoked by nuclear NCAM fragments without and with PSA indicates that PSA-carrying and -lacking NCAM play different functional roles in the nervous system.

LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription

MyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers.

Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome Capture.

Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.