Tracy K. Hale

The dynamic mobility of histone H1 is regulated by cyclin/CDK phosphorylation

ABSTRACT The linker histone H1 is involved in maintaining higher-order chromatin structures and displays dynamic nuclear mobility, which may be regulated by posttranslational modifications. To analyze the effect of H1 tail phosphorylation on the modulation of the histones nuclear dynamics, we generated a mutant histone H1, referred to as M1-5, in which the five cyclin-dependent kinase phosphorylation consensus sites were mutated from serine or threonine residues into alanines. Cyclin E/CDK2 or cyclin A/CDK2 cannot phosphorylate the mutant in vitro. Using the technique of fluorescence recovery after photobleaching, we observed that the mobility of a green fluorescent protein (GFP)-M1-5 fusion protein is decreased compared to that of a GFP-wild-type H1 fusion protein. In addition, recovery of H1 correlated with CDK2 activity, as GFP-H1 mobility was decreased in cells with low CDK2 activity. Blocking the activity of CDK2 by p21 expression decreased the mobility of GFP-H1 but not that of GFP-M1-5. Finally, the level and rate of recovery of cyan fluorescent protein (CFP)-M1-5 were lower than those of CFP-H1 specifically in heterochromatic regions. These data suggest that CDK2 phosphorylates histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions.

Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI/SNF or NURF.

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mousep53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of thep53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53promoter.

Helicases, G4-DNAs, and drug design.

New helicase assays that recognise therapeutically important G4‐DNA structures will lead to the discovery of novel molecular entities that bind not only to G4‐tetrads, but also to grooves and loops of G4‐DNA. Such assays can also provide inhibitors of G4‐specific helicases that will shed light on the emerging involvement of helicases in cancer and other diseases linked to defective DNA repair pathways.

PPM1B depletion induces premature senescence in human IMR-90 fibroblasts

p53 and NF-κB are key transcription factors in regulating the gene expression program of cellular and organismal senescence. PPM1B is a member of the protein phosphatase 2C family and plays a role in negatively regulating p53 and NF-κB thereby possibly attenuating the gene expression program of cellular senescence. Here, possible involvement of PPM1B in replicative senescence has been investigated using the in vitro aging model of IMR-90 cells. PPM1B protein levels are progressively decreased in a replicative age-dependent manner. Importantly, PPM1B depletion induces a robust senescence phenotype as evidenced by significant growth arrest and senescence marker expression. Given that PPM1B depletion-induced senescence is partially rescued by inactivating p38 MAPK, our results identify PPM1B as a critical regulator of both p38 MAPK-dependent and independent senescence pathways during normal cellular aging process.

Heterochromatin protein 1 expression is reduced in human thyroid malignancy

Owing to the loss of heterochromatin integrity that occurs during thyroid tumorigenesis, the expression of Heterochromatin Protein 1 isoforms HP1α and HP1β was assessed by immunohistochemistry in 189 thyroid tumors and non-neoplastic tissues. Expression of HP1β was significantly decreased in all thyroid lesions, except in follicular adenomas, when compared with matched adjacent normal tissue. This loss of HP1β expression may in part be caused by microRNA dysregulation. An example is miR-205, a microRNA that is abundantly upregulated in thyroid carcinomas and shown to reduce the expression of HP1β. In contrast to HP1β, HP1α expression was only reduced in metastatic carcinomas and poorly differentiated lesions. These results suggest the reduction of HP1β followed by a decrease in HP1α contributes to the pathogenesis of thyroid carcinomas, and their loss is a potential marker of thyroid malignancy and metastatic potential, respectively.

Abstract 1887: Untethering heterochromatin: how loss of HP1a enhances cell invasion by altering nuclear envelope integrity

Malignant cell invasion is accompanied by complex changes in nuclear shape and organisation. The onset of invasion correlates with the loss of HP1a in many solid tumours including those of the thyroid, kidney, colon and breast, while in cancer cell lines HP1a suppresses invasive potential. In normal cells HP1a maintains the condensation of transcriptionally silent heterochromatin and sequesters it at the inner periphery of the nuclear envelope; an event critical for the maintenance of nuclear stability and envelope rigidity. This role for HP1a leads us to hypothesise that the un-tethering of heterochromatin from the nuclear periphery that would occur after loss of HP1 causes a remodelling of nuclear envelope that enhances cellular invasion. If true this would explain how the nucleus, the largest and most rigid cellular organelles, becomes malleable enough to squeeze through interstitial spaces in the surrounding tissue during metastatic invasion. In support of this hypothesis we observed a 44% increase in the invasive potential of the poorly invasive MCF7 breast cells when HP1a is knocked down. Lamin A staining and electron microscopy also confirmed that these HP1a knockdown cells had the expected alterations in nuclear morphology and loss of heterochromatin at the nuclear periphery. Furthermore the loss of HP1 was correlated with a decrease in the mechanical stability of the nuclei. The significant enrichment (p-val 6.7x10 -8 ) of nuclear envelope proteins shown by RNAseq analysis of transcripts that are differentially expressed in the MCF7 versus HP1a knock-down cells, suggests this loss of nuclear envelope integrity is associated with alterations to its composition. The most striking change identified in the nuclear envelope of cells lacking HP1a was the loss of two Nesprin1 isoforms. While reintroduction of HP1a in highly invasive MDA-MB-231 cells shows the return of these isoforms to the nuclear envelope. As the Nesprin1 isoforms act as signalling scaffolds connecting to the cytoskeleton or reaching into the nucleus, they represent a means of modulating nuclear malleability. Together these findings suggest that when loss of HP1a increases invasive potential there is a corresponding disruption to the nuclear periphery that is a weakness of these cells that could be exploited. Citation Format: Tracy K. Hale, Sarah Bond, David Wheeler. Untethering heterochromatin: how loss of HP1a enhances cell invasion by altering nuclear envelope integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1887. doi:10.1158/1538-7445.AM2017-1887

Abstract 4538: Heterochromatin protein 1 alpha modulates the invasive potential of malignant cells

Altered chromatin patterning is observed in neoplastic cells, with loss of heterochromatin associated with aggressive malignancies. Heterochromatin is the tightly packed form of chromatin characterized by limited transcription and is identified by the localization of Heterochromatin Protein 1 alpha (HP1 alpha) and Heterochromatin Protein 1 beta (HP1 beta). HP1 alpha and HP1 beta help to maintain chromatin organization during the cell cycle and regulate gene expression. We have previously shown that HP1 alpha and HP1 beta are both downregulated in papillary thyroid carcinomas and that both proteins are lost to a greater degree in 3 out of 4 metastatic lesions of papillary thyroid carcinomas. In addition, others have shown a loss of HP1 alpha is associated with increased invasive potential and metastatic lesions in a range of human tumors. By modulating the level of HP1 alpha in breast cancer cell lines we demonstrate a causal role for HP1 alpha in regulating invasive potential. To identify the molecular pathways through which HP1 alpha regulates this phenotype, RNA-seq was used to measure changes in both miRNA and mRNA expression after knock-down of HP1 alpha in the poorly invasive breast cancer cell line, MCF7. Reduction of HP1 alpha levels increased their invasive potential by 44% and this corresponded with differential expression of genes that promote an epithelial to mesenchymal transition (EMT). In addition, a tissue microarray containing 56 invasive breast carcinomas was immunohistochemically stained for HP1 alpha and HP1 beta. A majority of the breast carcinomas showed decreased expression of HP1 alpha when compared to HP1 beta (32/56 tumors). Taken together, these data suggest that loss of HP1 alpha results in a phenotype that is an intermediate of the EMT, and is supported by their altered cytoskeleton, cell-cell and cell-matrix interactions. HP1 alpha loss may confer a more aggressive, invasive potential in breast carcinomas. By exploring these HP1 alpha −δeπeνδeντ pathways, we seek to further elucidate the regulatory signals that allow a malignant epithelial cell to undergo EMT and begin to invade surrounding tissue. Citation Format: Xiaoyan Cui, Lance Stimpson, Tracy Hale, Alejandro Contreras. Heterochromatin protein 1 alpha modulates the invasive potential of malignant cells. [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 4538.