Histone deacetylase (HDAC) 1 and 2 are Essential for normal T cell Development and Genomic Stability in Mice

Generation of T cell specific Hdac1 and Hdac2 Knock-out mice

Hdac1 and Hdac2 conditional knock-out alleles were generated using the targeting constructs previously described by Dovey et al, ²⁹ to target Hdac1 and Hdac2 loci in AB1.1;129S5 mouse embryonic stem cells using standard gene targeting methods. See Fig.S1 for detailed targeting strategy. Hdac1^Lox/Lox and Hdac2^Lox/Lox conditional knock-out mice, in which exon 2 has been flanked by LoxP sites, were inter-crossed with the transgenic Lck-Cre line ³⁰ to generate T cell specific Hdac1/2 deletion. These mice were further bred to the transgenic OT-II (Tg^OT-II-TCR) mice ³¹ to produce MHC class II restricted T cells. Genotyping primers are provided in Table.S2.

Isolation and flow cytometry analysis of lymphoid cells

Cells from thymus, spleen and lymph nodes were collected in ice chilled cell collection buffer (PBS, 3% fetal calf serum) and single cell suspensions generated. Cells were suspended in red blood cell lysis buffer (0.16M NH₄Cl, 0.01M KHCO₃), incubated at RT for 5 mins, pelleted and re-suspended in cell collection buffer. Cells for FACS were stained using the antibodies described in supplemental Table.S2. Cells were analysed using a BD FACSCanto II flow cytometer (BD Biosciences). FCS 3.0 files generated by FACS Diva software (BD Biosciences) were exported and analysed using VenturiOne (Applied Cytometry). Samples for mRNA extraction and whole genome expression profiling were stained as above and collected into cold PBS using a high speed MoFLo Legacy cell sorter (Beckman Coulter).

Protein and Enzymatic Analysis

To confirm loss of HDAC1 and HDAC2, cells were lysed with hypotonic buffer: 10mM HEPES pH7.9, 10mM KCl, 0.1mM EDTA and 10mM DTT. After centrifugation at 1,500g for 5 minutes nuclear extracts were prepared from pellets with: 20mM HEPES 7.9, 0.4M NaCl, 1mM EDTA and 25% glycerol. 10μg of nuclear extract was separated using SDS-PAGE and membranes probed with the appropriate antibodies (Table.S1). Acid extraction of histones was performed as previously described ²⁹. 5μg of extract was loaded in each lane and membranes probed using a panel of antibodies raised against a number of histone modifications. Membranes were scanned using the Odyssey Infrared imaging system and quantification of proteins achieved using the appropriate IRDye conjugated secondary antibodies (LiCOR Biosciences).

For immunoprecipitation, 70μg of nuclear extract was incubated overnight at 4°C with antibody coated protein-G agarose beads (GE Life Sciences). After 4 washes in nuclear extract buffer, beads were split into two aliquots. One aliquot was used to assess the deacetylase activity of the immunoprecipitates, while the second was resolved by SDS-PAGE and probed with antibodies raised against known components of the immunoprecipitated complexes.

Microarray and Comparative Genomic Hybridization analysis

Comparative gene expression profiles of stage matched (TCRβ^low/CD5^low) pre-lymphomic HD1^Δ2/Δ2; HD2^WT/Δ2 and wild type thymocytes were compared using an Illumina MouseWG-6 v2 Expression BeadChip platform (Illumina). Total RNA was isolated using a standard Trizol (Invitrogen) protocol and Phase Lock Gel Heavy Tubes (5 Prime). Quality control of total mRNA was performed using a 2100 Bioanalyser (Agilent). Only samples that had an RNA integrity number (RIN) of 8.6 or higher were selected for processing and array hybridisation. Processing of samples followed the manufacturer’s instructions. Detection P values <0.01 were used to filter all data. Significant differential expression between sample sets was defined as probes that exhibited a robust fold change of ≥2 (Fc≥2) with an adjusted P value of <0.005. An analysis of functionally related gene groups among deregulated genes and chromosomal distribution of deregulated genes was carried out using the Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7 ³².

DNA copy number variation in diseased thymocytes was assessed using the Mouse Genome Comparative Genomic Hybridisation 244K Microarray (Agilent Technologies) according to the manufacturer’s protocol. DNA was labelled with Cy3 or Cy5 according to BioPrime aCGH genomic labelling protocol (Invitrogen) and cleaned using purelink PCR purification kit (Invitrogen). Slides were hybridized for 48 hours, washed, and scanned with an Agilent microarray scanner. Raw data was then extracted using Agilent Feature Extraction. All following data analysis was performed in R using Bioconductor free packages (http://www.bioconductor.org), with background corrected then normalised using the loess algorithm.

HDAC1/2 are required for normal T cell development

To investigate the role of HDAC1/2 in T cell development we bred Hdac1^Lox/Lox and Hdac2^Lox/Lox conditional knock-out mice, in which exon 2 was flanked by LoxP sites (Fig.1A and Fig.S1), to the transgenic Lck-Cre line ³⁰. Southern blot and PCR analysis of DNA from Hdac1^{Lox/Lox; Lck-Cre} and Hdac2^{Lox/Lox; Lck-Cre} T cells revealed that Cre mediated deletion of exon 2 was >99% efficient, disrupting the ORF of both Hdac1 and Hdac2 such that a premature STOP codon is incorporated into exon 3, with a subsequent loss of protein (Fig.S2). Hdac1^{Lox/Lox; Lck-Cre} and Hdac2^{Lox/Lox; Lck-Cre} mice (henceforward referred to as Hdac1^Δ2/Δ2 and Hdac2^Δ2/Δ2) were then inter-crossed to generate all genotypic combinations: single (Hdac1^Δ2/Δ2, or Hdac2^Δ2/Δ2), compound (Hdac1^Δ2/Δ2; Hdac2^Δ2/WT, or Hdac1^Δ2/WT; Hdac2^Δ2/Δ2) and double (Hdac1&2^Δ2/Δ2) T cell specific knock-outs. T cell development can be delineated into a series of discrete steps by measuring the expression of the T cell receptor (TCR), its co-receptors CD4 and CD8, and CD5 using flow cytometric analysis (Fig.1B). The Lck-Cre transgene is active early, during the double-negative (DN) stage of thymopoiesis, allowing us to monitor HDAC1/2 function during each of these critical stages.

We began by analyzing Hdac1/2 deleted T cells from mice at 6-8 weeks of age (Fig.S3). Loss of either HDAC1, or HDAC2 alone produced no discernible phenotype with regards to the proportions of DN (CD4⁻CD8⁻), double positive (DP, CD4⁺CD8⁺), CD4 single-positive (CD4SP, CD4⁺CD8⁻) and CD8 single-positive (CD8SP, CD4⁻CD8⁺) T cells (Fig.S3B). Mice with a compound heterozygous/homozygous genotype, in which only a single copy of Hdac1 remains (Hdac1^Δ2/WT; Hdac2^Δ2/Δ2), also had a phenotype comparable to wild-type. However, in both Hdac1^Δ2/Δ2; Hdac2^Δ2/WT (bearing only a single copy of Hdac2) and Hdac1&2^Δ2/Δ2 mice, we observed a significant increase in CD4^Low/CD8^High cells, including a 4-fold increase in the percentage of immature single positive cells compared to wild type (ISP, CD4^Int/CD8⁺ - 4% versus 18% and 17% respectively), indicating a block in development. Despite a strong phenotypic effect, we consistently observed near wild-type levels of HDAC2 protein in double knock-out mice (Fig.S3A, arrow), presumably because of selective pressure over time, due to the essential requirement for HDAC1/2 during T cell development. In an attempt to overcome this we switched our analysis to the neonatal stage (10-14 days), because at earlier time-points we observed a more robust, simultaneous, loss of HDAC1 and HDAC2 protein (Fig.1C). Neonatal Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice again display a significant increase in CD4^low/CD8^high cells (Fig.1C, 4% to 51%). We also observed a 2-fold reduction in the percentage of mature TCRβ^high/CD5^high thymocytes (10% to 4%) and a 3-fold increase in TCRβ^Int/CD5^Low cells (8% to 25%), identifying this population as developmentally immature. The absolute number of CD4^Low/CD8^High cells is increased 10-fold, while DP cells are reduced by 2.6-fold, indicating a block in DN to DP transition due to Hdac1/2 haploinsufficiency (Fig.S4A). This also leads to increased levels of apoptosis in CD4^Low/CD8^High and DP cells in both neonates and 6-week old mice (Fig.S4B).

Uniquely among all the genotypes tested, Hdac1&2^Δ2/Δ2 mice had a marked 5-fold reduction in thymocyte cellularity (Fig.1D), with decreases in the number of mature CD4SP and CD8SP cells (Fig.1E). This reduction is likely related to the increased percentage of DN cells in Hdac1&2^Δ2/Δ2 mice (Fig.1C, 3% to 27%). However, the absolute number of DN cells was similar in mice of all genotypes (Fig.1F, left panel), as were the relative populations of DN1, DN2, DN3 and DN4 cells (adjudged by CD25/CD44 staining, right panel). The DN to DP transition involves a proliferative burst, which may be impaired due to a generic requirement for HDAC1/2 during cell cycle progression, similar to that observed in other Hdac1/2 knock-out systems ^1,33,34.

HDAC1/2 deficient T cells exhibit positive selection defects and reduced CD4 lineage commitment

To prevent defective proliferation from confounding analysis of the developmental phenotypes, we focused our analysis on the two compound heterozygote/homozygote genotypes, Hdac1^Δ2/WT; Hdac2^Δ2/Δ2 and Hdac1^Δ2/Δ2; Hdac2^Δ2/WT, in comparison with wild-type mice. Staining for TCRβ expression in Hdac1^Δ2/Δ2; Hdac2^Δ2/WT cells (which retain a single copy of Hdac2), but not Hdac1^Δ2/WT; Hdac2^Δ2/Δ2 (single copy of Hdac1), reveals a significant increase in developmentally immature cells, at a level of TCRβ consistent with DP cells (Fig.2A, right panel). In order to progress from the DP intermediate stage to CD4SP (helper), or CD8SP (cytotoxic) lineages, thymocytes must undergo positive selection mediated by TCR/MHC interactions, or die from neglect. Staining for CD69, which is transiently expressed in cells undergoing positive selection, in conjunction with TCRβ staining, permits fractionation of individual developmental sub-populations, namely: immature (TCRβ^low/CD69^low), pre-selection (TCRβ^int/CD69^low), thymocytes undergoing selection (TCRβ^int/CD69^int), and post-selection (TCRβ^high/CD69^high) cells. The proportion of positively selected thymocytes is reduced 2-fold, and post-selection thymocytes 3-fold (Fig.2B), in cells with a single copy of Hdac2 (Hdac1^Δ2/Δ2; Hdac2^Δ2/WT). Gating on TCRβ^int/CD69^int cells undergoing positive selection, we observed very few CD4^high/CD8^low cells, which are primed for terminal differentiation into CD4 or CD8 SP thymocytes (Fig.2B, lower panels). Hdac1^Δ2/WT; Hdac2^Δ2/Δ2 mice (single copy HDAC1) display a more subtle reduction in positively-selected cells.

To address whether this phenotype was TCR dependent we inter-crossed Hdac1^Δ2/Δ2; Hdac2^Δ2/WT with OT-II (Tg^OT-II-TCR) mice which express pre-rearranged transgenic forms of TCRα and TCRβ. The resulting Hdac1^Δ2/Δ2; Hdac2^Δ2/WT-Tg^OT-II mice help to test two hypotheses. Firstly, if defects in positive-selection are due to a non-functional TCR the presence of the Tg^OT-II-TCR should rescue the cellularity of mature T cells; and secondly, as the TCR produced by the Tg^OT-II-TCR transgene recognises only MHC class-II molecules (generating only CD4SP cells), this might be expected to counteract the bias towards development of CD8-positive cells in Hdac1/2 deleted T cells. Thus, CD4/CD8 co-receptor expression was analyzed on neonatal thymocytes from WT and Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice in the presence or absence of the Tg^OT-II-TCR transgene (Fig.2C). As expected, the majority of SP cells in WT-Tg^OT-II-TCR mice were CD4SP. However, the presence of the pre-arranged TCR failed to rescue the CD8-phenotype in Hdac1^Δ2/Δ2; Hdac2^Δ2/WT-Tg^OT-II-TCR thymocytes. Furthermore, the absolute number of mature SP thymocytes (CD4SP or CD8SP) was not significantly changed on the Tg^OT-II-TCR background (Fig.3D). These data indicate that the lack of positive selection and the accumulation of CD4^low/CD8^high cells cannot be rescued by the presence of an intact rearranged TCR complex.

Loss of co-repressor complex integrity and increased histone acetylation in Hdac1/2 knock-out thymocytes

To address the biochemical mechanism underlying the block in T cell development, we measured the enzymatic activity of HDAC1/2 containing co-repressor complexes, and levels of histone acetylation in knock-out cells. Consistent with previous reports ^13,35, deletion of Hdac1 (Hdac1^Δ2/Δ2) results in a 2-fold increase in the level of HDAC2 protein (Fig.3A and Fig.S5). Significantly, a 50% reduction in Hdac2 gene dosage reduces this compensatory increase in HDAC2 protein (compare Hdac1^Δ2/Δ2 and Hdac1^Δ2/Δ2; Hdac2^Δ2/WT, centre panel), potentially explaining the difference in T cell phenotype observed between these two genotypes (Fig.1C). In contrast, deletion of Hdac2 (Hdac2^Δ2/Δ2) does not affect HDAC1 protein levels. Hdac1&2^Δ2/Δ2 mice were not used in these analyses due to the severe reduction in neonatal cellularity and compensatory increase in HDAC2 levels at later time points.

We began by measuring the total deacetylase activity of nuclei isolated from single and compound Hdac1/2 knock-out T cells. Loss of Hdac2 alone had no overall effect (Fig.3B). In contrast, deletion of Hdac2 and a single copy of Hdac1 (Hdac1^Δ2/WT; Hdac2^Δ2/Δ2), Hdac1 alone, or Hdac1 and a single copy of Hdac2 (Hdac1^Δ2/Δ2; Hdac2^Δ2/WT), reduced total deacetylase activity significantly. Even with a single WT Hdac2 allele remaining in Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice, HDAC1/2 accounted for at least 60% of total deacetylase activity, making them the predominant HDAC enzymes in the T cell nucleus. Within the nucleus they form the catalytic components of corepressor complexes, principally, Sin3A, NuRD and CoREST ³⁶. By immunoprecipitating Sin3A and MTA2 (a major component of NuRD) we were able to measure the residual deacetylase activity of the Sin3A and NuRD complexes in the absence of HDAC1/2 (Fig.3B, 3C). Deletion of Hdac1 alone, but not Hdac2, results in a reduction of deacetylase activity associated with both Sin3A and NuRD complexes. Interestingly, Sin3A deacetylase activity was unaltered in Hdac1^Δ2/WT; Hdac2^Δ2/Δ2 compound mice, which correlates well with the almost complete absence of HDAC2 from the Sin3A complex in WT cells (Fig.3C, compare lanes 3 and 11). In Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice, Sin3A activity was reduced 2-fold and NuRD 5-fold, prompting us test the integrity of complex components. Unexpectedly, we found that levels of both Sin3A and MTA2 protein, both of which interact with HDAC1/2 directly, were significantly reduced (Fig.3A). Whereas, Mi2β and LSD1, which are thought to be recruited to the NuRD and CoREST complexes independently of HDAC1/2, were unaffected.

Since individual HDAC1/2 complexes were perturbed, we proceeded to measure levels of global histone acetylation (Fig.3D). Consistent with the loss of deacetylase activity, we observed a 3-fold increase in histone H3 K9 acetylation (H3K9Ac) in Hdac^Δ2/Δ2; Hdac2^Δ2/WT cells. Global levels of histone H3 K14, K27 and H4 K12 acetylation were also increased significantly (Fig.S5C), although to a lesser degree than H3K9Ac. In contrast, H3 K9 tri-methylation (H3K9me3) a marker of heterochromatic chromatin, which is largely devoid of HDAC1/2, was unaffected by deletion of either protein.

HDAC1/2 regulate the T cell transcriptome

The alteration in histone acetylation in Hdac1/2 deleted cells (Fig.3D) is likely to affect a change in the gene expression program, potentially leading to the observed block in T cell development. To analyze the transcriptome in Hdac1/2 deleted cells, thymocytes from 2-3 week old WT (n=7) and Hdac1^Δ2/Δ2; Hdac2^Δ2/WT (n=5) mice, in which cell number was unaffected (Fig.4A), were isolated, and RNA extracted from FACS sorted pre-selection populations (TCRβ^low/CD5^low). Comparative microarray analysis was performed and transcripts de-regulated ≥2-fold (Fc ≥2, adjusted P<0.005) identified. Hierarchical clustering based on signal detection values of >8,000 genes revealed different transcriptional programs in WT vs. Hdac1^Δ2/Δ2; Hdac2^Δ2/WT thymocytes (Fig.4A, right panel). A total of 5,954 transcripts were detected in either genotype, with 892 de-regulated (Fig.4B and Supplemental Information for complete gene list). Consistent with a role for HDAC1/2 in transcriptional repression more transcripts are up-regulated (542), than down-regulated (350). An analysis of functionally related gene groups among de-regulated genes using DAVID ³², reveals that genes involved in chromatin organization/assembly, DNA replication and T cell activation/differentiation were among the top 10 significantly enriched gene clusters (Fig.4B). Performing the same analysis on separate up- and down-regulated gene lists identifies genes involved in DNA replication are up-regulated, conversely, genes assigned to T cell activation/differentiation are down-regulated (Fig.4C). The down-regulated cluster includes a number of genes involved in V(D)J recombination (Rag1, Rag2 and DCLRE1C the gene that encodes Artemis). This suggests TCR recombination has been completed in (TCRβ^low/CD5^low) Hdac1^Δ2/Δ2; Hdac2^Δ2/WT thymocytes, but is still ongoing in WT DP cells which have yet to rearrange their TCRα chain. Consistent with the developmental block, and lack of selection thymocytes (TCRβ^int/CD69^int), Cd69 is identified as down-regulated as are key mediators of T cell signalling such as LAT (linker of activated T cells), Themis (thymocyte expressed molecule involved in selection), Itk (IL-2 inducible T cell kinase) and Tec (Tec protein-tyrosine kinase). Down-regulation of these genes suggests a model in which the absence of HDAC1/2 results in defective propagation of TCR signalling, thus blocking development.

Loss of HDAC1/2 results in a lethal pathology as a result of intra-thymic T cell neoplastic transformation

During the analysis it became apparent that a high proportion of Hdac1^Δ2/Δ2; Hdac2^Δ2/WT and Hdac1&2^Δ2/Δ2 mice become moribund by 15 weeks of age, typified by an enlarged chest or abdomen and rapid respiration. We therefore monitored the survival of cohorts of all genotypes over a five month period (Fig.5A). Lethality of Hdac1^Δ2/Δ2; Hdac2^Δ2/WT and Hdac1&2^Δ2/Δ2 mice occurred with 100% penetrance at an average of 12- and 15-weeks respectively. The lag of 3-weeks in median survival of Hdac1&2^Δ2/Δ2 mice likely reflects the time taken to overcome the 5-fold reduction in thymocyte cellularity at the neonatal stage (Fig.1C, 1D). By six weeks of age all Hdac1&2^Δ2/Δ2 mice have undergone selection for cells which retain near wild-type levels of HDAC2 (Fig.S3C), and thus tumour cells from double knock-out mice effectively phenocopy the Hdac1^Δ2/Δ2; Hdac2^Δ2/WT genotype. Tumour incidence correlates with cell types containing the lowest HDAC activity (Fig.3B and 5A). It is interesting to note therefore that one of eight HDAC1^Δ2/Δ2 mice also developed a tumor, suggesting that the level of HDAC activity in these cells is close to the threshold required for normal T cell development. Necropsy of these mice revealed development of a pathological diseased state characterized by massively enlarged thymus and splenomegaly (Fig.5B). FACS analysis of cells from these tissues reveals that they are overwhelmingly populated by immature (TCRβ^int/CD5^Int) CD4^low/CD8^high and DP T cells (Fig.5C and 5D), which exhibited a side- and forward-scatter profile characteristic of a ‘blasting’ state, indicative of cellular proliferation (Fig.S6A). Histological analysis confirms that the thymus and spleen are inundated with proliferative (Ki67 positive) T cells, with complete effacement of medullary and cortical sub-structures in the diseased thymus (Fig.5E). Infiltration of T cells into non-lymphatic tissue (lung, liver and kidney) in 6 of 14 Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice examined was also detectable (Fig.5F). This aggressive and lethal increase in thymocytes could occur either as a consequence of a general hyperplasia (related to the developmental block) or expansion of cells with a monoclonal neoplastic transformation. To distinguish between these two possibilities we used PCR to determine the Dβ1-Jβ1 and Dβ2-Jβ2 rearrangements at the TCRβ locus. If the increase in cellularity was a consequence of general hyperplasia we would expect to detect multiple recombined events (similar to WT, Fig.5G, lanes 2, 4 and 6), representative of a diverse and polyclonal population. In fact, thymocytes from 6-8 week Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice exhibited oligo-clonal TCRβ rearrangements which become increasingly monoclonal in diseased mice of the same genotype (Fig.5G, 2/4 samples have a single rearranged PCR fragment, lanes 11 and 14); indicating monoclonal expansion rather than a general hyperproliferation of immature T cells. Thus, in addition to a block in T cell development, Hdac1/2 haploinsufficiency in T cells results in neoplastic transformation and tumorigenesis.

Tumorigenic Hdac1^Δ2/Δ2;Hdac2^Δ2/WT T cells are associated with chromosomal instability

Chromosomal instability is a hallmark of human tumors ³⁷ and has been shown to play a critical role in the initiation and progression of haematological malignancies ³⁸. We therefore used array comparative genomic hybridization (aCGH) to detect gain or loss of genetic material in lymphomas derived from Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice (n=6). All of the tumors displayed aneuploidy, with 4/6 containing trisomy 15 and the remainder (2/6) trisomy 2 (Fig.6A and Fig.S6 for complete data series). There were 16 additional unique focal gains and deletions, including one sample with a homozygous deletion of the tumor suppressor Pten. Consistent with results assessing clonality (Fig.5G), all tumor samples revealed deletions at the Tcra (chromosome 14) and Tcrb loci (chromosome 6). Trisomy of chromosome 15 is characteristic of mouse T cell tumors ^39,40 and is associated with the up-regulation of the proto-oncogene, c-Myc (located on chromosome 15 in mice). In agreement with array data (which show a 2.5-fold increase in c-Myc), western blotting reveals elevation of c-Myc expression in 5/6 tumors when compared to WT controls (Fig.6B) including all samples with trisomy 15. Although gene dosage may contribute to the increase in c-Myc protein, we also observed elevated c-Myc levels in a tumor sample wild-type for chromosome 15 (tumor 6, Fig.S6), which suggests that additional factors may also play a role.

Chromosomal instability associated with tumor progression is generally coincident with elevated levels of DNA damage and a genome-wide loss of DNA methylation. HDAC1/2 have been suggested to stabilise Dnmt1 protein levels ⁴¹; however, we found no difference in Dnmt1, or Dnmt3b, and little or no change in the DNA-methylation status of genomic repeats (Fig.S7). HDAC1/2 also play a role in the DNA damage response (DDR) pathway, deacetylating chromatin in the vicinity of double strand breaks ⁴². Strikingly, we observed a 4-fold increase in γH2AX levels in Hdac1^Δ2/Δ2; Hdac2^Δ2/WT thymocytes compared to controls, indicative of substantial DNA damage in HDAC1/2 deleted cells (Fig.6C). Acetyl-lysines in both histone H3 (H3K56ac) and H4 (H4K16ac) are deacetylated in order to facilitate DNA repair ^42,43. Hdac1^Δ2/Δ2; Hdac2^Δ2/WT thymocytes display a 1.5 and 2.1-fold increase in global levels of H3K56Ac and H4K16Ac respectively, suggesting that these sites are regulated directly by HDAC1/2 containing complexes. These data suggest that HDAC1/2 play a crucial role in maintaining genomic stability independently of global DNA methylation status.

HDAC1/2 and co-repressor complex integrity are required for normal T cell development

We have demonstrated an essential role for HDAC1 and 2 (HDAC1/2) in the establishment of mature T cell populations. Double homozygous deletion of Hdac1/2 (Hdac1&2^Δ2/Δ2) in the T cells of neonatal mice, results in a 5-fold reduction in total thymocyte cellularity (Fig.1D) and a block in DN to DP transition (Fig.1E). Reduced cell numbers correlate with a significant increase in the percentage of DN cells (although absolute numbers of DN cells remains unchanged), indicating a block in DN to DP transition. The DN-DP transition is marked not only by expression of the CD4/CD8 co-receptors, but also by proliferative expansion; thus defects in cell cycle at this stage of development would explain the subsequent decrease in DP, CD4SP and CD8SP populations. Loss of both HDAC1/2 causes a severe cell cycle block in a number of systems, due to defects in gene regulation (e.g. Cdk inhibitors p21 and p57) ^34,35,44 and mitosis ¹. Conditional ablation of mSin3a in T cells results in a similar increase in DN cells, and a failure to proliferate in response to TCR signalling, suggesting that the DN to DP transition is mediated via the intact Sin3A:HDAC1/2 complex ²⁶.

Our analysis of mice with compound Hdac1/2 genotypes reveals a partial biochemical redundancy between the two enzymes. Retention of a single Hdac1 allele in the absence of Hdac2 (Hdac1^Δ2/WT; Hdac2^Δ2/Δ2) is less deleterious, in terms of total deacetylase activity and phenotype, than deletion of Hdac1 alone, or Hdac1 and a single copy of Hdac2 (Hdac1^Δ2/Δ2; Hdac2^Δ2/WT, Fig.3B). In agreement with our findings, a single Hdac1 allele, but not Hdac2, is sufficient to ensure normal B cell differentiation. ³⁶. This, along with data from embryonic stem cells ²⁹, suggests that HDAC1 is the more dominant of the two enzymes. Removal of HDAC1/2 from the Sin3A and NuRD complexes causes increased protein turnover (mRNA levels are unaffected) of the direct HDAC binding subunits Sin3A and MTA2 respectively (Fig.3A), a phenomenon also observed in compound HDAC1/2 knock-out ES cells (unpublished observations). An X-ray crystal structure of HDAC1 complexed with MTA1 reveals extensive interactions between HDAC1 and the adjacent ELM2/SANT domains (J. Schwabe – personal communication). The ELM2 domain in particular contains an extended loop that wraps around HDAC1, which in the absence of the enzyme is highly unlikely to be folded, and therefore lead to increased proteolysis. This is of particular importance when considering the lymphoma that occurs in Hdac1/2 deleted mice (Fig.5, discussed below) and the outcome of patients taking HDAC-inhibitors (HDACi) for the treatment of haematological malignancies. Our data indicate that HDAC1/2 provide a structural role, in addition to their enzymatic function, within the Sin3A and NuRD co-repressor complexes; and thus the absence of the protein generates a much more pronounced phenotype in mice than inhibition of the same enzymes in situ with HDACi.

HDAC1/2 are required for the development of CD4SP cells

The reduction of deacetylase activity associated with Hdac1/2 deletion in T cells (Hdac1^Δ2/Δ2; Hdac2^Δ2/WT > Hdac1^Δ2/Δ2 > Hdac1^Δ2/WT; Hdac2^Δ2/Δ2) correlates with a developmental block, in which immature CD4^Low/CD8^High and DP cells accumulate and fail to undergo positive selection (Fig.1C, 2B). Expression of an MHC class-II restricted TCR did not rescue the bias towards CD8-positive cells, and Cd4 transcription was up-regulated in Hdac1^Δ2/Δ2; Hdac2^Δ2/WT cells (Fig.4B and supplemental data), indicating a direct effect of HDAC1/2 on Cd4 gene expression (Fig.2C, 4C). Conditional inactivation of Mi2β, a central component of the NuRD complex and an ATP-dependent chromatin remodeler, results in a developmental phenotype with strikingly similar characteristics to Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice, that is, accumulation of DP-like thymocytes which fail to express CD4 ²⁷. Mi2β is thought to activate CD4 expression in DP cells independently of HDAC1/2 (and therefore NuRD), by recruiting histone acetyltransferase enzymes, p300 and MOZ, to the Cd4 silencer, thus antagonizing the repressive function of Ikaros. We can assert with confidence that Mi2β potentiates Cd4 transcription (monitored using a Cd4 promoter/silencer transgene) and that deletion of Mi2β causes loss of histone acetylation across the first intron, which encompasses the silencer element ⁴⁵. However, given the similarity of the Mi2β and Hdac1/2 knock-out phenotypes and perturbation of the complex by down-regulation of the MTA2 sub-unit, we suggest that Cd4 activation is a canonical NuRD dependent process. The classical notion of HDAC1/2 as exclusive mediators of transcriptional repression has been recently challenged by a Chip-Seq study (using peripheral CD4SP lymphocytes) which demonstrates that HDAC1/2 are predominantly located in the vicinity of active promoters, coincident with histone hyper-acetylation ¹¹. Furthermore, HDAC1/2 activity can be negatively regulated via acetylation by p300 ⁴⁶ (and potentially MOZ), which in the context of the NuRD complex would leave the chromatin remodelling activity of Mi2β as the dominant enzymatic function, and thus potentially able to function as a co-activator at the Cd4 silencer.

HDAC1/2 maintain global histone acetylation and chromosomal stability in developing thymocytes

All lymphomas isolated from Hdac1/2 deleted mice exhibited chromosomal instability (Fig.6A and Fig.S6B). This is consistent with previous reports which have shown that HDAC activity is required for faithful chromosome segregation and maintenance of constitutive heterochromatin through subsequent cell divisions ^47-49. The significance of this result in our system is that it occurs on a haploinsufficient (Hdac1^Δ2/Δ2; Hdac2^Δ2/WT) background. Indeed, Hdac1&2^Δ2/Δ2 mice by 6 weeks have age have begun to for select cells which have failed to inactivate Hdac2, and therefore phenocopy Hdac1^Δ2/Δ2; Hdac2^Δ2/WT mice (Fig.S3). Complete loss of HDAC1/2 causes cell cycle arrest in numerous tissue systems ^1,33,44,50, therefore retention of some HDAC1/2 activity, albeit at a significantly reduced level (Fig.3B), allows cells to divide while perturbing accurate chromosome segregation. This may have significant bearing for the use of HDAC1/2 as potential therapeutic targets in the treatment of cancer. Complete inhibition of HDAC1/2 activity will cause tumor cells to arrest or undergo apoptosis ⁵⁰, while partial inhibition might result in the opposite of the desired effect.

We have demonstrated an essential role for HDAC1/2 in the development of mature T cell populations. Deletion of Hdac1/2 causes a marked reduction in thymocytes cellularity and a block in the DN-DP transition, most likely due to the down-regulation of key TCR signalling components. We have also shown, for the first time in a physiological system, that HDAC1/2 activity is critical for maintaining genome stability. The occurrence of tumors (and the developmental block) is dose dependent, occurring in cells with the least amount of deacetylase activity, indicating that regulation of the acetyl-proteome, a balance between acetyltransferase and deacetylase enzymes, is crucial for normal cell development and viability.