Histone deacetylase 1 plays a predominant pro-oncogenic role in Eµ-myc driven B cell lymphoma.

The two histone deacetylases (Hdacs), Hdac1 and Hdac2, are erasers of acetylation marks on histone tails, and are important regulators of gene expression that were shown to play important roles in hematological malignancies. However, several recent studies reported opposing tumor-suppressive or tumor-promoting roles for Hdac1 and Hdac2. Here, we investigated the functional role of Hdac1 and Hdac2 using the Eµ-myc mouse model of B cell lymphoma. We demonstrate that Hdac1 and Hdac2 have a pro-oncogenic role in both Eµ-myc tumorigenesis and tumor maintenance. Hdac1 and Hdac2 promote tumorigenesis in a gene dose-dependent manner, with a predominant function of Hdac1. Our data show that Hdac1 and Hdac2 impact on Eµ-myc B cell proliferation and apoptosis and suggest that a critical level of Hdac activity may be required for Eµ-myc tumorigenesis and proper B cell development. This provides the rationale for utilization of selective Hdac1 and Hdac2 inhibitors in the treatment of hematological malignancies.

Hematopoietic overexpression of FOG1 does not affect B-cells but reduces the number of circulating eosinophils.

We have identified expression of the gene encoding the transcriptional coactivator FOG-1 (Friend of GATA-1; Zfpm1, Zinc finger protein multitype 1) in B lymphocytes. We found that FOG-1 expression is directly or indirectly dependent on the B cell-specific coactivator OBF-1 and that it is modulated during B cell development: expression is observed in early but not in late stages of B cell development. To directly test in vivo the role of FOG-1 in B lymphocytes, we developed a novel embryonic stem cell recombination system. For this, we combined homologous recombination with the FLP recombinase activity to rapidly generate embryonic stem cell lines carrying a Cre-inducible transgene at the Rosa26 locus. Using this system, we successfully generated transgenic mice where FOG-1 is conditionally overexpressed in mature B-cells or in the entire hematopoietic system. While overexpression of FOG-1 in B cells did not significantly affect B cell development or function, we found that enforced expression of FOG-1 throughout all hematopoietic lineages led to a reduction in the number of circulating eosinophils, confirming and extending to mammals the known function of FOG-1 in this lineage.

Histone deacetylase 6 (HDAC6) is an essential modifier of glucocorticoid-induced hepatic gluconeogenesis.

In the current study, we investigated the importance of histone deacetylase (HDAC)6 for glucocorticoid receptor-mediated effects on glucose metabolism and its potential as a therapeutic target for the prevention of glucocorticoid-induced diabetes. Dexamethasone-induced hepatic glucose output and glucocorticoid receptor translocation were analyzed in wild-type (wt) and HDAC6-deficient (HDAC6KO) mice. The effect of the specific HDAC6 inhibitor tubacin was analyzed in vitro. wt and HDAC6KO mice were subjected to 3 weeks' dexamethasone treatment before analysis of glucose and insulin tolerance. HDAC6KO mice showed impaired dexamethasone-induced hepatic glucocorticoid receptor translocation. Accordingly, dexamethasone-induced expression of a large number of hepatic genes was significantly attenuated in mice lacking HDAC6 and by tubacin in vitro. Glucose output of primary hepatocytes from HDAC6KO mice was diminished. A significant improvement of dexamethasone-induced whole-body glucose intolerance as well as insulin resistance in HDAC6KO mice compared with wt littermates was observed. This study demonstrates that HDAC6 is an essential regulator of hepatic glucocorticoid-stimulated gluconeogenesis and impairment of whole-body glucose metabolism through modification of glucocorticoid receptor nuclear translocation. Selective pharmacological inhibition of HDAC6 may provide a future therapeutic option against the prodiabetogenic actions of glucocorticoids.

Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

Enforced expression of the transcriptional coactivator OBF1 impairs B cell differentiation at the earliest stage of development.

OBF1, also known as Bob.1 or OCA-B, is a B lymphocyte-specific transcription factor which coactivates Oct1 and Oct2 on B cell specific promoters. So far, the function of OBF1 has been mainly identified in late stage B cell populations. The central defect of OBF1 deficient mice is a severely reduced immune response to T cell-dependent antigens and a lack of germinal center formation in the spleen. Relatively little is known about a potential function of OBF1 in developing B cells. Here we have generated transgenic mice overexpressing OBF1 in B cells under the control of the immunoglobulin heavy chain promoter and enhancer. Surprisingly, these mice have greatly reduced numbers of follicular B cells in the periphery and have a compromised immune response. Furthermore, B cell differentiation is impaired at an early stage in the bone marrow: a first block is observed during B cell commitment and a second differentiation block is seen at the large preB2 cell stage. The cells that succeed to escape the block and to differentiate into mature B cells have post-translationally downregulated the expression of transgene, indicating that expression of OBF1 beyond the normal level early in B cell development is deleterious. Transcriptome analysis identified genes deregulated in these mice and Id2 and Id3, two known negative regulators of B cell differentiation, were found to be upregulated in the EPLM and preB cells of the transgenic mice. Furthermore, the Id2 and Id3 promoters contain octamer-like sites, to which OBF1 can bind. These results provide evidence that tight regulation of OBF1 expression in early B cells is essential to allow efficient B lymphocyte differentiation.

Silencing and nuclear repositioning of the lambda5 gene locus at the pre-B cell stage requires Aiolos and OBF-1.

The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes lambda5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the lambda5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally.

Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, alpha-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.

The OBF-1 gene locus confers B cell-specific transcription by restricting the ubiquitous activity of its promoter.

Transcription of the gene encoding the transcriptional coactivator Oct-binding factor 1 (OBF-1)/OCA-B/Bob.1 is largely restricted to B cells. During B cell development OBF-1 expression shows two peaks, one in immature B cells in the bone marrow and the other in germinal center B cells. Promoter analysis has identified a cAMP response element (CRE)-binding site present in the OBF-1 proximal promoter that is crucial for activity in B cells and for the induction of OBF-1 expression upon stimulation with CD40 ligand/IL-4. Here we address the question of how transcription of the OBF-1 gene is restricted to B cells. Surprisingly, in transient transfection assays the OBF-1 proximal promoter exhibited an equally strong activity in B and non-B cells. In contrast, upstream promoter regions displayed B cell-specific properties, partly overlapping with DNaseI hypersensitive sites identified in this study. In mice, expression of a neomycin resistance gene under the control of a Polyoma enhancer/TK promoter cassette was restricted to B cells when integrated into the OBF-1 locus, but was ubiquitous when integrated into two other loci, Oct-1 or the large subunit of RNA polymerase II.Therefore, lineage commitment of the OBF-1 gene is promoter independent and is achieved by regulating the entire locus in a B cell-specific manner.

HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo.

Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes. Reversible acetylation on the epsilon-amino group of alpha-tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics. Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently. Here we report that beta-tubulin interacts with histone deacetylase-6 (HDAC-6) in a yeast two-hybrid assay and in vitro. We find that HDAC-6 is a micro tubule-associated protein capable of deacetylating alpha-tubulin in vivo and in vitro. HDAC-6's microtubule binding and deacetylation functions both depend on the hdac domains. Overexpression of HDAC-6 in mammalian cells leads to tubulin hypoacetylation. In contrast, inhibition of HDAC-6 function by two independent mechanisms--pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC-6 in embryonic stem cells)--leads to hyperacetylation of tubulin and microtubules. Taken together, our data provide evidence that HDAC-6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non-histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors.

Lack of the transcriptional coactivator OBF-1 prevents the development of systemic lupus erythematosus-like phenotypes in Aiolos mutant mice.

Here we show that mice lacking the zinc finger transcription factor Aiolos develop the symptoms of human systemic lupus erythematosus (SLE), which is characterized by the production of anti-dsDNA Ab and immune complex-mediated glomerulonephritis. This finding indicates that normal Aiolos function is necessary to maintain immune homeostasis and suppress the development of systemic autoimmune disease and implicates Aiolos as a possible candidate gene for SLE. Interestingly, Aiolos-null mice can no longer mount autoimmune reactions and completely fail to develop SLE when they are deficient for the B cell-specific transcription coactivator OBF-1. The lack of OBF-1 reverses several Aiolos mutant mouse phenotypes, such as B cell hyperproliferation, high expression of activation marker on B cells, and spontaneous germinal center formation. Unexpectedly, B cell development at the immature B cell stage is severely impaired in the bone marrow of Aiolos/OBF-1 double-deficient mice, demonstrating the key role of these factors in the transition from pre-B to immature B cells. Our results indicate that B cells play a crucial role in the development of SLE in Aiolos mutant mice and might be useful for the strategy of SLE treatment.