Diminished phosphorylation of a heat shock protein (HSP 27) in infant acute lymphoblastic leukemia

SummaryWe have previously reported lack of expression of a polypeptide designated L3 in infant acute lymphoblastic leukemia (ALL). Expression of L3 occurred predominantly in older children with pre-B ALL. We have recently reported the expression during B cell ontogeny of two other polypeptides, designated L2 and L4 with a similar Mr as L3, which were identified as phosphorylated and non-phosphorylated forms respectively of the low Mr heat shock protein, hsp27. In this study we have characterized L3 and identified it as another phosphorylated form of hsp27. The two phosphorylated forms appear to be differentially expressed in acute leukemia. L3 levels in infants who expressed hsp27 isoforms L2 and L4 were significantly diminished compared to levels in older children with an equivalent amount of hsp27. We conclude that leukemic cells in infant ALL exhibit a unique pattern of phosphorylation of hsp27 expressed at a pre-B cell stage of differentiation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29447/1/0000529.pd

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

Functional diversity of histone H1 variants may be caused by differences in phosphorylation during interphase

The Transcription Factor Ultraspiracle Influences Honey Bee Social Behavior and Behavior-Related Gene Expression

Behavior is among the most dynamic animal phenotypes, modulated by a variety of internal and external stimuli. Behavioral differences are associated with large-scale changes in gene expression, but little is known about how these changes are regulated. Here we show how a transcription factor (TF), ultraspiracle (usp; the insect homolog of the Retinoid X Receptor), working in complex transcriptional networks, can regulate behavioral plasticity and associated changes in gene expression. We first show that RNAi knockdown of USP in honey bee abdominal fat bodies delayed the transition from working in the hive (primarily “nursing” brood) to foraging outside. We then demonstrate through transcriptomics experiments that USP induced many maturation-related transcriptional changes in the fat bodies by mediating transcriptional responses to juvenile hormone. These maturation-related transcriptional responses to USP occurred without changes in USP's genomic binding sites, as revealed by ChIP–chip. Instead, behaviorally related gene expression is likely determined by combinatorial interactions between USP and other TFs whose cis-regulatory motifs were enriched at USP's binding sites. Many modules of JH– and maturation-related genes were co-regulated in both the fat body and brain, predicting that usp and cofactors influence shared transcriptional networks in both of these maturation-related tissues. Our findings demonstrate how “single gene effects” on behavioral plasticity can involve complex transcriptional networks, in both brain and peripheral tissues

Genetic and epigenetic variation in histone H1

grantor: University of TorontoHistone proteins play a fundamental role in DNA-templated processes in eukaryotes. Access of regulatory factors for DNA replication, repair and gene transcription, to binding sites in chromatin is influenced by histone binding to DNA in nucleosomes, and by higher order folding of nucleosomal filaments that is mediated, in part, by histone H1. Non-allelic amino acid sequence variants of histone H1 are co-expressed in most eukaryotes, and tissue-specific patterns of H1 variant expression in metazoans have long suggested that H1 variants may be functionally distinct. However, direct support for this hypothesis is lacking, in part, due to the inability of current chromatographic procedures to adequately resolve H1 variants and post-translationally modified forms. To facilitate investigation of the functional significance of H1 heterogeneity, a novel HPLC method, cation-exchange-hydrophilic-interaction chromatography (CX-HILIC), was developed that resolved 14 major and numerous minor peaks for H1 from pooled chicken blood. This was unexpected because only six genes for H1 variants are known in this species. Electrophoretic analyses revealed that each CX-HILIC peak represented one of the six non-allelic variants of H1 described previously, but that four variants were represented by multiple peaks. Mass spectrometry revealed that HI recovered from CX-HILIC peaks was more heterogenous than was apparent in electrophoretic analyses and that the molecular mass of most molecules was smaller than predicted by the corresponding gene sequences. This was not attributable to proteolysis during handling since CX-HILIC peaks of H5 processed in parallel contained molecules that matched the expected value or exceeded it due to covalent modification. Analyses of H1 prepared from blood of individual chickens revealed that allelic variation was responsible for much of the CX-HILIC heterogeneity of H1 from pooled blood. Analyses of peptides from pooled erythrocyte H1 and liver H1 suggested that heterogeneity also arises from limited proteolysis of N-termini of H1 in vivo. Amino acid sequencing revealed that the genes encoding two non-allelic variants have been identified incorrectly in the literature. Together, these analyses demonstrate an unexpectedly high degree of heterogeneity in avian H1 and suggest the novel hypothesis that limited proteolysis may regulate H1 function in vivo.Ph.D

Site-specific regulation of histone H1 phosphorylation in pluripotent cell differentiation

Abstract Background Structural variation among histone H1 variants confers distinct modes of chromatin binding that are important for differential regulation of chromatin condensation, gene expression and other processes. Changes in the expression and genomic distributions of H1 variants during cell differentiation appear to contribute to phenotypic differences between cell types, but few details are known about the roles of individual H1 variants and the significance of their disparate capacities for phosphorylation. In this study, we investigated the dynamics of interphase phosphorylation at specific sites in individual H1 variants during the differentiation of pluripotent NT2 and mouse embryonic stem cells and characterized the kinases involved in regulating specific H1 variant phosphorylations in NT2 and HeLa cells. Results Here, we show that the global levels of phosphorylation at H1.5-Ser18 (pS18-H1.5), H1.2/H1.5-Ser173 (pS173-H1.2/5) and H1.4-Ser187 (pS187-H1.4) are regulated differentially during pluripotent cell differentiation. Enrichment of pS187-H1.4 near the transcription start site of pluripotency factor genes in pluripotent cells is markedly reduced upon differentiation, whereas pS187-H1.4 levels at housekeeping genes are largely unaltered. Selective inhibition of CDK7 or CDK9 rapidly diminishes pS187-H1.4 levels globally and its enrichment at housekeeping genes, and similar responses were observed following depletion of CDK9. These data suggest that H1.4-S187 is a bona fide substrate for CDK9, a notion that is further supported by the significant colocalization of CDK9 and pS187-H1.4 to gene promoters in reciprocal re-ChIP analyses. Moreover, treating cells with actinomycin D to inhibit transcription and trigger the release of active CDK9/P-TEFb from 7SK snRNA complexes induces the accumulation of pS187-H1.4 at promoters and gene bodies. Notably, the levels of pS187-H1.4 enrichment after actinomycin D treatment or cell differentiation reflect the extent of CDK9 recruitment at the same loci. Remarkably, the global levels of H1.5-S18 and H1.2/H1.5-S173 phosphorylation are not affected by these transcription inhibitor treatments, and selective inhibition of CDK2 does not affect the global levels of phosphorylation at H1.4-S187 or H1.5-S18. Conclusions Our data provide strong evidence that H1 variant interphase phosphorylation is dynamically regulated in a site-specific and gene-specific fashion during pluripotent cell differentiation, and that enrichment of pS187-H1.4 at genes is positively related to their transcription. H1.4-S187 is likely to be a direct target of CDK9 during interphase, suggesting the possibility that this particular phosphorylation may contribute to the release of paused RNA pol II. In contrast, the other H1 variant phosphorylations we investigated appear to be mediated by distinct kinases and further analyses are needed to determine their functional significance

MOESM1 of Site-specific regulation of histone H1 phosphorylation in pluripotent cell differentiation

Additional file 1: Table S1. List of ChIP-qPCR primers

MOESM2 of Site-specific regulation of histone H1 phosphorylation in pluripotent cell differentiation

Additional file 2: Figure S1. Validations of custom H1 antibodies that have not been published previously. A, Recombinant H1 variants were analyzed by immunoblotting with our custom antisera for H1.5 and H1.0. B, PCA-extracted crude H1 from WI-38 VA-13 cells and a mixture of recombinant H1.5 and H1.0 were analyzed by immunoblotting with our custom antisera against pS18-H1.5. C, Antisera against pS18-H1.5 was mock-treated (none) or preadsorbed with pS18-H1.5 antigen peptide (pS18) or the corresponding non-phosphorylated peptide (S18) prior to immunoblotting with HeLa whole-cell lysate. D, HeLa and WI-38 VA-13 whole-cell lysates (WCL) and PCA-extracted crude H1 (PCAS) from WI-38 VA-13 cells were analyzed by immunoblotting with our custom antisera against H1.5 and H1.0