Shihua He

Nuclear organization and chromatin dynamics--Sp1, Sp3 and histone deacetylases.

Regulation of gene expression involves the coordinated activities and interplay between chromatin remodeling factors and transcription factor recruitment. Histone acetyltransferases, histone deacetylases, histone kinases, histone phosphatases, histone methyltransferases, histone demethylases and ATP-dependent chromatin remodeling complexes mediate chromatin remodeling and are components of a complex epigenetic network regulating gene expression during development and differentiation. Transcription factors play key roles in the recruitment of histone modifying enzymes and chromatin remodeling complexes to specific gene promoters. Sp1 and Sp3 are two transcription factors that are expressed in all mammalian cells and are involved in the regulation of genes involved in most cellular processes. Remodeling of chromatin is a necessary event in preparing the gene for transcription. In this review we will cover the organization and remodeling of chromatin, with a focus on dynamic histone acetylation and the histone deacetylase enzymes. The structure and function of transcription factors Sp1 and Sp3 will be presented. The role of these factors in the regulation of the estrogen responsive trefoil factor 1 gene will be highlighted. In the analyses of the factors involved in the regulation of the expression of a specific gene, the chromatin immunoprecipitation assay in which the protein factor of interest is cross-linked to DNA with formaldehyde is an essential tool. The limitations of this assay in cancer cells in which genomic instability is rampant are discussed.

Chromatin organization and nuclear microenvironments in cancer cells

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. However, our knowledge of events resulting in changes in nuclear shape and chromatin organization in cancer cells is limited. Nuclear matrix proteins, which include lamins, transcription factors (Sp1) and histone modifying enzymes (histone deacetylases), and histone modifications (histone H3 phosphorylation) have roles in organizing chromatin in the interphase nucleus, regulating gene expression programs and determining nuclear shape. Histone H3 phosphorylation, a downstream target of the Ras‐mitogen activated protein kinase pathway, is involved in neoplastic transformation. This article will review genetic and epigenetic events that alter chromatin organization in cancer cells and the role of the nuclear matrix in determining nuclear morphology. J. Cell. Biochem. 104: 2004–2015, 2008.

Chromatin Modification of the Trefoil Factor 1 Gene in Human Breast Cancer Cells by the Ras/Mitogen-Activated Protein Kinase Pathway

Histone H3 phosphorylation is a downstream response to activation of the Ras/mitogen-activated protein kinase (MAPK) pathway. This modification is thought to have a role in chromatin remodeling and in the initiation of gene transcription. In MCF-7 breast cancer cells, we observed that phosphorylated histone H3 (phospho-H3) at Ser(10) but not Ser(28) increased with phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) treatment. Although phosphorylated extracellular signal-regulated kinase 1/2 levels in these cells cultured under estradiol deplete and replete conditions displayed no change, a significant induction was observed after TPA treatment. Furthermore, whereas both estradiol and TPA increased trefoil factor 1 (TFF1) mRNA levels in these cells, only TPA-induced and not estradiol-induced TFF1 expression was inhibited by the H3 kinase mitogen and stress activated protein kinase (MSK) inhibitor H89 and MAPK kinase inhibitor UO126, showing the involvement of the Ras/MAPK following TPA induction. Mutation of the activator protein 1 (AP-1) binding site abrogated the TPA-induced transcriptional response of the luciferase reporter gene under the control of the TFF1 promoter, showing the requirement for the AP-1 site. In chromatin immunoprecipitation assays, estradiol treatment resulted in the association of the estrogen receptor-alpha (ERalpha) and acetylated H3 with the TFF1 promoter. The levels of phospho-H3 and MSK1 associated with the TFF1 promoter were moderately increased. In the presence of TPA, whereas ERalpha was not bound to the promoter, a strong association of acetylated and/or phospho-H3, MSK1, and c-Jun was observed. These results show that although both stimuli lead to TFF1 gene activation, estradiol and TPA exert their effects on TFF1 gene expression by different mechanisms.

Mitotic partitioning of transcription factors

Mitosis is a highly orchestrated process involving numerous protein kinases and phosphatases. At the onset of mitosis, the chromatin condensation into metaphase chromosomes is correlated with global phosphorylation of histone H3. The bulk of transcription is silenced while many of the transcription‐associated proteins, including transcription and chromatin remodeling factors, are excluded from chromatin, typically as a consequence of their phosphorylation. Components of the transcription machinery and regulatory proteins are recycled and equally partitioned between newly divided cells by mechanisms that may involve microtubules, microfilaments or intermediate filaments. However, as demonstrated in the case of Runx2, a subset of transcription factors involved in lineage‐specific control, likely remain associated with their target genes to direct the deposition or removal of epigenetic marks. The displacement and re‐entry into daughter cells of transcription and chromatin remodeling factors are temporally defined and regulated. Reformation of daughter nuclei is a critical time to re‐establish the proper gene expression pattern. The mechanisms involved in the marking and re‐establishment of gene expression has been elucidated for few genes. The elucidation of how the memory of a programmed expression profile is transmitted to daughter cells represents a challenge. J. Cell. Biochem. 105: 1–8, 2008.

Histone H3 phosphorylation, immediate-early gene expression, and the nucleosomal response: a historical perspective.

Histone H3 is modified at serines 10 and 28 in interphase cells following activation of the RAS-MAPK or p38-MAPK pathways by growth factors or stress. These modifications are involved in the regulation of immediate-early genes, including Jun and Fos, whose increased expression is a trademark of various cancers. This review outlines the series of discoveries that led to the characterization of these modifications, the kinase, MSK1/2, which is activated by both MAPK pathways and directs phosphorylation of H3, and the mechanistic function of these modifications in transcriptional activation. Research examining the effect of deregulated MSK1/2 in human disorders, namely cancer, is evaluated. Recently, a number of reports proposed novel, intervening pathways leading to enrichment of phosphorylated serine 10 and 28 and the activation of MSK1/2. These novel pathways predict an even more complicated signalling mechanism for cell growth, apoptosis, and the immune response, suggesting that MSK1/2 is intrinsically responsible for an even greater number of biological processes. This review proposes that MSK1/2 is an optimal target for cancer therapy, based on its fundamental role in transmitting external signals into varied responses involved in cancer development.

Sp1 and Sp3 foci distribution throughout mitosis.

The mammalian transcription factors Sp1 and Sp3 compete for the same DNA binding sites but play different roles in the regulation of expression of numerous genes. It is known that, in the interphase nucleus, Sp1 and Sp3 are organized into distinct foci. In this study, we show that throughout the mitotic process, while being displaced from the condensed chromosomes and dispersed throughout the cell, Sp1 and Sp3 maintain their separate punctate distributions. In metaphase, both Sp1 and Sp3 foci show a high degree of colocalization with microfilaments, suggesting that F-actin is involved in the organization of Sp1 and Sp3 foci during mitosis. Constant Sp1 and Sp3 levels were observed during mitosis, signifying a recovery of the pre-existing Sp1 and Sp3 population in newly formed nuclei. In late telophase, Sp1 and Sp3 are equally segregated between daughter cells, and their subnuclear organization as distinct foci is restored in a sequential fashion with Sp3 regrouping into the newly formed nuclei prior to Sp1. Both Sp1 and Sp3 return to the nuclei ahead of RNA polymerase II. Our results support a model in which entry of Sp1, Sp3 and RNA polymerase II into the newly formed nuclei is an ordered process.

Role of MSK1 in the Malignant Phenotype of Ras-transformed Mouse Fibroblasts

Activated by the RAS-MAPK signaling pathway, MSK1 is recruited to immediate-early gene (IEG) regulatory regions, where it phosphorylates histone H3 at Ser-10 or Ser-28. Chromatin remodelers and modifiers are then recruited by 14-3-3 proteins, readers of phosphoserine marks, leading to the occupancy of IEG promoters by the initiation-engaged form of RNA polymerase II and the onset of transcription. In this study, we show that this mechanism of IEG induction, initially elucidated in parental 10T1/2 murine fibroblast cells, applies to metastatic Hras1-transformed Ciras-3 cells. As the RAS-MAPK pathway is constitutively activated in Ciras-3 cells, MSK1 activity and phosphorylated H3 steady-state levels are elevated. We found that steady-state levels of the IEG products AP-1 and COX-2 were also elevated in Ciras-3 cells. When MSK1 activity was inhibited or MSK1 expression was knocked down in Ciras-3 cells, the induction of IEG expression and the steady-state levels of COX-2, FRA-1, and JUN were greatly reduced. Furthermore, MSK1 knockdown Ciras-3 cells lost their malignant phenotype, as reflected by the absence of anchorage-independent growth.

Selective Association of Peroxiredoxin 1 with Genomic DNA and COX-2 Upstream Promoter Elements in Estrogen Receptor Negative Breast Cancer Cells

PRDX1 was identified as a protein preferentially crosslinked to DNA in estrogen receptor negative but not in estrogen receptor positive breast cancer cells. In estrogen receptor negative cells, PRDX1 is phosphorylated, binds to NF-κB, and is recruited to COX-2 upstream promoter elements.

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Background: HDAC1 and -2 homo- or heterodimers within corepressor complexes are displaced from chromatin during mitosis. Results: CK2-mediated mitotic phosphorylated HDAC1 and -2 are dissociated from each other but not from corepressor complexes. Conclusion: HDAC1 or HDAC2 homodimers, but not heterodimers, are responsible for the activity of mitotic corepressor complexes. Significance: Mitotically phosphorylated HDAC1 and -2 potentially target different cellular proteins. Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.

Dynamic distribution of HDAC1 and HDAC2 during mitosis: Association with F-actin

During mitosis, histone deacetylase 2 (HDAC2) becomes highly phosphorylated through the action of CK2, and HDAC1 and 2 are displaced from mitotic chromosomes. HDAC1 and 2 are components of corepressor complexes, which function with lysine acetyltransferases to catalyze dynamic protein acetylation and regulate gene expression. In this study, we show that HDAC1 and 2 associate with F‐actin in mitotic cells. Inhibition of Aurora B or protein kinase CK2 did not prevent the displacement of HDAC1 and 2 from mitotic chromosomes in HeLa cells. Further, proteins of the HDAC1 and 2 corepressor complexes and transcription factors recruiting these corepressors to chromatin were dissociated from mitotic chromosomes independent of Aurora B activity. HDAC1 and 2 returned to the nuclei of daughter cells during lamin A/C reassembly and before Sp1, Sp3, and RNA polymerase II. Our results show that HDAC1 and 2 corepressor complexes are removed from the mitotic chromosomes and are available early in the events leading to the re‐establishment of the gene expression program in daughter cells. J. Cell. Physiol. 228: 1525–1535, 2013.