Essential role for histone deacetylase 11 (HDAC11) in neutrophil biology.

Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL-10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage-restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.

The Tumor Suppressive Effects of HPP1 Are Mediated Through JAK-STAT-Interferon Signaling Pathways.

HPP1, a novel tumor suppressive epidermal growth factor (EGF)-like ligand, mediates its effects through signal transducer and activators of transcription (STAT) activation. We previously demonstrated the importance of STAT1 activation for HPP1 function; however the contribution of STAT2 remains unclear. We sought to delineate the components of JAK-STAT-interferon (IFN) signaling specifically associated with HPP1s biological effects. Using stable HPP1-HCT116 transfectants, expression analyses were performed by polymerase chain reaction (PCR)/western blotting while expression knockdowns were achieved using siRNA. Growth parameters evaluated included proliferation, cell cycle distribution, and anchorage-independent growth. STAT dimerization, translocation, and DNA binding were examined by reporter assays, fluorescent microscopy, and chromatin immunoprecipitation (ChIP), respectively. Forced expression of HPP1 in colon cancer cell lines results in the upregulation of total and activated levels of STAT2. We have also determined that JAK1 and JAK2 are activated in response to HPP1 overexpression, and are necessary for subsequent STAT activation. Overexpression of HPP1 was associated with significant increases in STAT1:STAT1 (p=0.007) and STAT1:STAT2 (p=0.036) dimer formation, as well as subsequent nuclear translocation. By ChIP, binding of activated STAT1 and STAT2 to the interferon-signaling regulatory element promoter sites of the selected genes, protein kinase RNA-activated (PKR), IFI44, and OAS1 was demonstrated. STAT2 knockdown resulted in partial abrogation of HPP1s growth suppressive activity with increased proliferation (p<0.0001), reduced G1/G0 phase cell cycle fraction, and a restoration of growth potential in soft agar (p<0.01). Presumably as a consequence of upregulation of IFN signaling elements, HPP1 overexpression resulted in an acquisition of exogenous IFN sensitivity. Physiologic doses of IFN-α resulted in a significant reduction in proliferation (p<0.001) and increase in G1/G0 cell cycle arrest in HPP1 transfectants. STAT2 is necessary for HPP1-associated growth suppression, and mediates these effects through activation of IFN-α pathways. Given the interest in therapeutic targeting of oncogenic erbB proteins, further understanding of HPP1s role as a tumor suppressive EGF-like ligand is warranted.

MDM2 interaction with nuclear corepressor KAP1 contributes to p53 inactivation.

MDM2 is a RING domain ubiquitin E3 ligase and a major regulator of the p53 tumor suppressor. MDM2 binds to p53, inactivates p53 transcription function, inhibits p53 acetylation, and promotes p53 degradation. Here, we present evidence that MDM2 interacts with the nuclear corepressor KAP1. The binding is mediated by the N-terminal coiled-coil domain of KAP1 and the central acidic domain of MDM2. KAP1 stimulates formation of p53-HDAC1 complex and inhibits p53 acetylation by interacting with MDM2. Expression of KAP1 cooperates with MDM2 to promote p53 ubiquitination and degradation. The tumor suppressor ARF competes with KAP1 in MDM2 binding; oncogene induction of ARF expression reduces MDM2-KAP1 interaction. Depletion of endogenous KAP1 expression by RNAi stimulates p53 transcriptional activity, sensitizes p53 response to DNA damage, and increases apoptosis. Therefore, MDM2 interaction with KAP1 contributes to p53 functional regulation. ARF may regulate p53 acetylation and stability in part by inhibiting KAP1-MDM2 binding.

Identification of a functionally impaired positive regulatory domain I binding factor 1 transcription repressor in myeloma cell lines.

B cell differentiation into a plasma cell requires expression of the positive regulatory domain zinc finger protein 1 gene (PRDM1) that encodes the positive regulatory domain I binding factor 1 (PRDI-BF1 or Blimp-1) protein. It represses the transcription of specific target genes, including c-myc, the MHC class II trans-activator, Pax-5, and CD23b. In this study we demonstrate the presence of an alternative protein product of the PRDM1 gene. The new protein, PRDI-BF1 beta, has a disrupted PR domain and lacks the amino-terminal 101 aa of the originally described protein. PRDI-BF1 beta has a dramatic loss of repressive function on multiple target genes, but maintains normal DNA-binding activity, nuclear localization, and association with histone deacetylases and deacetylase activity. Myeloma cell lines express the highest levels of PRDM1 beta mRNA relative to the full-length form, while primary cells and several other cell lines have very low, but detectable, levels of PRDM1 beta. RNA analysis and analysis of the PRDM1 promoters demonstrate that PRDI-BF1 beta is generated from the same gene by alternative transcription initiation using an internal promoter. These newly described features of the PRDM1 gene are highly analogous to the PRDM2 (RIZ) and PRDM3 (MDS1-EVI1) genes, in which each express a truncated protein missing the PR domain. The expression of each of the truncated proteins is elevated in cancerous cells and may play an important role in the disease.