The hematopoietic transcription factor GATA-1 regulates erythropoiesis and ␤-globin expression. Although consensus GATA-1 binding sites exist throughout the murine ␤-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the ␤-globin promoters. The hematopoietic factor NF-E2 cooperated with GATA-1 to recruit pol II to the promoters. We propose that only when GATA-1 attracts pol II to the locus control region can pol II access the promoter in a NF-E2-dependent manner. E ukaryotic DNA wraps Ϸ1.7 times around a core histone octamer to form nucleosomes, which in turn, fold into highly condensed chromatin. The role of chromatin structure in regulating gene expression is established through extensive analysis in diverse systems (1-8). An important consequence of chromatin folding is the regulation of cis-element accessibility, thereby preventing the constitutive loading of trans-acting factors and RNA polymerase II (pol II). Coactivators and corepressors are recruited to chromosomal sites via interactions with transcription factors and catalyze chromatin remodeling (9, 10). Although certain factors can recognize binding sites on nucleosomal DNA, others are occluded (11,12). Factors capable of binding nucleosomal sites would not likely be able to access sites in condensed chromatin. Thus, site occupancy in vivo cannot be predicted by sequence analysis, but rather requires analysis of binding in living cells.We use the murine ␤-globin locus to investigate how transcriptional control occurs within chromatin domains (13,14). The ␤-globin locus consists of several genes arrayed in the order of their developmental expression (15). High-level transcription of these genes requires an upstream locus control region (LCR) (16-19), comprised of four DNaseI hypersensitive sites, 21). Besides an erythroid-specific enhancer function, the LCR counteracts transgene silencing (16,22).A common feature of active chromatin is core histone acetylation (23). We hypothesized that LCRs function by recruiting histone acetyltransferases that establish broad acetylation patterns (24). Analysis of the human GH domain in transgenic mice has provided strong evidence for the hypothesis that LCRs can establish broad acetylation patterns (25). Analysis of acetylation at the murine ␤-globin locus in adult erythroid cells revealed enrichment of acetylated histones H3 and H4 at the LCR and the adult globin genes, ␤major and ␤minor (24, 26). Much less acetylation was evident over a Ϸ30-kb region spanning the silent embryonic ␤-globin genes Ey and ␤H1, and between the adult ␤-globin genes. In embryonic erythroid cells, acetylation was high at the LCR, the embryonic ␤H1 promoter and the inactive ␤minor promoter (24). Although the LCR confers high-level ␤-globin expression, deletion of HS1-HS4 from the murine locus did not abrogate hyperacetylatio...

Posttranslational modification of histones through acetylation, methylation, and phosphorylation is a common mode of regulating chromatin structure and, therefore, diverse nuclear processes. One such modification, methylated histone H3 at lysine-4 (H3-meK4), colocalizes with hyperacetylated histones H3 and H4 in mammalian chromatin. Whereas activators directly recruit acetyltransferases, the process whereby H3-meK4 is established is unknown. We tested whether the hematopoietic-specific activators NF-E2 and GATA-1, which mediate transactivation of the ␤-globin genes, induce both histone acetylation and H3-meK4. Through the use of NF-E2-and GATA-1-null cell lines, we show that both activators induce H3 acetylation at the promoter upon transcriptional activation. However, analysis of H3-mek4 revealed that NF-E2 and GATA-1 differentially regulate chromatin modifications at the ␤major promoter. NF-E2, but not GATA-1, induces H3-meK4 at the promoter. Thus, under conditions in which NF-E2 and GATA-1 activate the transcription of an endogenous gene at least 570-fold, these activators differ in their capacity to induce H3-meK4. Despite strong H3-meK4 at hypersensitive site 2 of the upstream locus control region, neither factor was required to establish H3-meK4 at this site. These results support a model in which multiple tissuespecific activators collectively function to assemble a composite histone modification pattern, consisting of overlapping histone acetylation and methylation. As GATA-1 induced H3 acetylation, but not H3-meK4, at the promoter, H3 acetylation and H3-meK4 components of a composite histone modification pattern can be established independently.globin ͉ chromosome ͉ erythroid ͉ epigenetic C hromatin structure at the promoters and over broad chromosomal segments is a critical determinant of gene expression, and therefore, chromatin modification is an essential step in transcriptional control. The acetylation of core histones in nucleosomes represents a major mode of chromatin modification (1-3). Histone acetylation increases the accessibility of nucleosomal DNA through structural transitions at the level of the nucleosome (4, 5) and higher-order chromatin structure (6). In addition to directly modifying chromatin structure, the acetyllysine residue can be recognized by regulatory factors, e.g., bromodomain-containing coactivators (7-9). Such coactivators catalyze additional chromatin modification and interact with components of the transcriptional machinery, thereby stimulating transcription (10).Besides acetylation, histones are subjected to methylation, phosphorylation, and ubiquitination (11). Given the combinatorial complexity of histone modifications, a ''histone code'' hypothesis has been proposed, which assumes that distinct patterns of modifications confer unique functional consequences (12). For example, distinct methyltransferases methylate histone H3 at either lysine-4 (H3-meK4) (13-15) or lysine-9 (H3-meK9) (16), and the distribution of H3-meK4 and H3-meK9 delineates functionally unique chrom...

Certain class II MHC (MHCII) alleles in mice and humans confer risk for or protection from type 1 diabetes (T1D). Insulin is a major autoantigen in T1D, but how its peptides are presented to CD4 T cells by MHCII risk alleles has been controversial. In the mouse model of T1D, CD4 T cells respond to insulin B-chain peptide (B:9-23) mimotopes engineered to bind the mouse MHCII molecule, IA g7 , in an unfavorable position or register. Because of the similarities between IA g7 and human HLA-DQ T1D risk alleles, we examined control and T1D subjects with these risk alleles for CD4 T-cell responses to the same natural B:9-23 peptide and mimotopes. A high proportion of new-onset T1D subjects mounted an inflammatory IFN-γ response much more frequently to one of the mimotope peptides than to the natural peptide. Surprisingly, the control subjects bearing an HLA-DQ risk allele also did. However, these control subjects, especially those with only one HLA-DQ risk allele, very frequently made an IL-10 response, a cytokine associated with regulatory T cells. T1D subjects with established disease also responded to the mimotope rather than the natural B:9-23 peptide in proliferation assays and the proliferating cells were highly enriched in certain T-cell receptor sequences. Our results suggest that the risk of T1D may be related to how an HLA-DQ genotype determines the balance of T-cell inflammatory vs. regulatory responses to insulin, having important implications for the use and monitoring of insulinspecific therapies to prevent diabetes onset.T ype 1 diabetes (T1D), the autoimmune form of diabetes, results from T cell-mediated destruction of insulin-producing β-cells within pancreatic islets (1). The disease is dramatically increasing in incidence, doubling in the last two decades (2, 3), and now predictable with the measurement of antibodies directed against insulin and other proteins found in β-cells (4). Major efforts at disease prevention have been undertaken using preparations of insulin (s.c., oral, and intranasal) to induce tolerance and delay the onset of clinical symptoms (5-7). Measuring insulin-specific T-cell responses from the peripheral blood has been challenging but would allow for assessment of therapeutic response, which has been a major obstacle in these trials. In our study, we sought to detect peripheral T-cell responses to insulin in T1D patients and nondiabetic controls using a modified insulin B-chain peptide.Insulin is a major self-antigen for both T and B cells in murine and human T1D, with insulin B-chain amino acids 9-23 (B:9-23) being a key epitope presented by major histocompatibility complex class II (MHCII) molecules to CD4 T cells targeting pancreatic β-cells (8-10). There is strong evidence from the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes that the NOD MHCII molecule, IA g7 , is required for development of T1D and that pathogenic CD4 T cells recognize insulin B:9-23 presented in an unfavorable position or register (Reg3) in the IA g7 peptide binding groove (9, 11,...

Post-translational modifications of individual lysine residues of core histones can exert unique functional consequences. For example, methylation of histone H3 at lysine 79 (H3-meK79) has been implicated recently in gene silencing in Saccharomyces cerevisiae. However, the distribution and function of H3-meK79 in mammalian chromatin are not known. We found that H3-meK79 has a variable distribution within the murine ␤-globin locus in adult erythroid cells, being preferentially enriched at the active ␤major gene. By contrast, acetylated H3 and H4 and H3 methylated at lysine 4 were enriched both at ␤major and at the upstream locus control region. H3-meK79 was also enriched at the active cad gene, whereas the transcriptionally inactive loci necdin and MyoD1 contained very little H3-meK79. As the pattern of H3-meK79 at the ␤-globin locus differed between adult and embryonic erythroid cells, establishment and/or maintenance of H3-meK79 was developmentally dynamic. Genetic complementation analysis in null cells lacking the erythroid and megakaryocyte-specific transcription factor p45/NF-E2 showed that p45/NF-E2 preferentially establishes H3-meK79 at the ␤major promoter. These results support a model in which H3-meK79 is strongly enriched in mammalian chromatin at active genes but not uniformly throughout active chromatin domains. As H3-meK79 is highly regulated at the ␤-globin locus, we propose that the murine ortholog of Disruptor of Telomeric Silencing-1-like (mDOT1L) methyltransferase, which synthesizes H3-meK79, regulates ␤-globin transcription.Post-translational modification of core histones in chromatin represents a common epigenetic mechanism that controls diverse nuclear processes. An almost endless number of histone modifications has been documented, and it has been hypothesized that a histone code exists in which unique combinations of histone modifications confer distinct functional consequences (1). Acetylation of core histones enhances factor access to the chromatin template by increasing accessibility of nucleosomal DNA (2, 3). Increased accessibility does not appear to be accompanied by a major change in nucleosome structure in vitro (4, 5). However, a 3-fold increase in acetylation strongly inhibits folding of chromatin fibers into higher order structures (6). Furthermore, the acetylated amino-terminal tails of core histones are recognized by bromodomains (7,8), which are often present in coactivators that mediate transcriptional activation. Although the structure of acetylated chromatin and the functional implications of acetylation have been studied for many years (9), considerably less is known about other histone modifications such as methylation.The concept of a histone code is illustrated by methylation of histone H3 at amino acids 4 and 9. Histone H3 methylated at lysine 9 (H3-meK9), 1 but not at lysine 4 (H3-meK4), is selectively recognized by heterochromatin protein 1 (HP1) (10 -14). HP1 serves an important role in the assembly of repressive chromatin structures (15), thus linking a site-specif...

Although dialysis has been used in the care of patients with acute kidney injury (AKI) for over 50 years, very little is known about the potential benefits of uremic control on systemic complications of AKI. Since the mortality of AKI requiring renal replacement therapy (RRT) is greater than half in the intensive care unit a better understanding of the potential of RRT to improve outcomes is urgently needed. Therefore, we sought to develop a technically feasible and reproducible model of RRT in a mouse model of AKI. Models of low and high dose peritoneal dialysis (PD) were developed and their effect on AKI, systemic inflammation, and lung injury after ischemic AKI was examined. High dose PD had no effect on AKI, but effectively cleared serum IL-6, and dramatically reduced lung inflammation while low dose PD had no effect on any of these three outcomes. Both models of RRT using PD in AKI in mice reliably lowered urea in a dose dependent fashion. Thus, use of these models of PD in mice with AKI has great potential to unravel the mechanisms by which RRT may improve the systemic complications that have led to increased mortality in AKI. In light of recent data demonstrating reduced serum IL-6 and improved outcomes with prophylactic PD in children, we believe our results are highly clinically relevant.

Although it is well established that acute kidney injury (AKI) is a proinflammatory state, little is known about the endogenous counter-inflammatory response. IL-6 is traditionally considered a pro-inflammatory cytokine that is elevated in the serum in both human and murine AKI. However, IL-6 is known to have anti-inflammatory effects. Here we sought to investigate the role of IL-6 in the counter-inflammatory response after AKI, particularly in regard to the anti-inflammatory cytokine IL-10. Ischemic AKI was induced by bilateral renal pedicle clamping. IL-10-deficient mice had increased systemic and lung inflammation after AKI, demonstrating the role of IL-10 in limiting inflammation after AKI. We then sought to determine whether IL-6 mediates IL-10 production. Wild-type mice with AKI had a marked upregulation of splenic IL-10 that was absent in IL-6-deficient mice with AKI. In vitro, addition of IL-6 to splenocytes increased IL-10 production in CD4 T cells, B cells, and macrophages. In vivo, CD4-deficient mice with AKI had reduced splenic IL-10 and increased lung myeloperoxidase activity. Thus, IL-6 directly increases IL-10 production and participates in the counter-inflammatory response after AKI.

Histone H3 methylated at lysine 4 (H3-meK4) co-localizes with hyperacetylated histones H3 and H4 in transcriptionally active chromatin, but mechanisms that establish H3-meK4 are poorly understood. Previously, we showed that the hematopoietic-specific activator NF-E2, which is required for ␤-globin transcription in erythroleukemia cells, induces histone H3 hyperacetylation and H3-meK4 at the adult ␤-globin genes (␤major and ␤minor). Chromatin immunoprecipitation analysis indicated that NF-E2 occupies hypersensitive site two (HS2) of the ␤-globin locus control region. The mechanism of NF-E2-mediated chromatin modification was investigated by complementation analysis in NF-E2-null CB3 erythroleukemia cells. The activation domain of the hematopoietic-specific subunit of NF-E2 (p45/NF-E2) contains two WW domainbinding motifs (PXY-1 and PXY-2). PXY-1 is required for activation of ␤-globin transcription. Here, we determined which step in NF-E2-dependent transactivation is PXY-1-dependent. A p45/NF-E2 mutant lacking 42 amino acids of the activation domain, including both PXY motifs, and a mutant lacking only PXY-1 were impaired in inducing histone H3 hyperacetylation, H3-meK4, and RNA polymerase II recruitment. The PXY motifs were not required for transactivation in the context of a GAL4 DNA-binding domain fusion to p45/NF-E2 in transient transfection assays. As the PXY-1 mutant occupied HS2 normally, the chromatin modification defect occurred post-DNA binding. PXY-1 was not required for recruitment of the histone acetyltransferases cAMP-responsive elementbinding protein-binding protein (CBP) and p300 to HS2. These results indicate that PXY-1 confers chromatinspecific transcriptional activation via interaction with a co-regulator distinct from CBP/p300 or by regulating CBP/p300 function.Mechanisms that dynamically regulate chromatin structure at localized sites and over broad chromosomal regions are crucial for controlling nuclear processes such as transcription. A common mode of regulating chromatin structure is the posttranslational modification of core histones, of which histone acetylation is the most extensively studied (1, 2). Hyperacetylated histones are often enriched in transcriptionally active chromatin (3, 4), although reductions in acetylation can occur upon transcriptional activation (5). Acetylation impacts transcription in part by increasing DNA accessibility within the nucleosome (6) and by perturbation of higher order chromatin folding (7,8).Analogous to acetylation, methylation of lysine 4 of histone H3 (H3-meK4) 1 marks transcriptionally active chromatin (9 -13). By contrast, histone H3 methylated at lysine 9 is associated with transcriptional repression (14 -16). At least one mechanism by which H3-meK4 functions is by inhibiting binding of the nucleosome remodeling deacetylase repressor complex to the amino-terminal tail of histone H3, thereby favoring the transcriptionally active state (17). Acetylated and methylated lysines within histones can also function as ligands to bind bromodomain-and chromo...

SummaryType 1 diabetes (T1D) is an autoimmune disease that destroys the insulinproducing beta-islet cells of the pancreas. Currently, there are no treatment modalities for prevention of T1D, and the mechanisms influencing disease inception and early progression are not well understood. We have used the insulin 2 -/-non-obese diabetic (Ins2 -/-NOD) model to study stages of T1D and to examine the protective effects of a potent analogue of 1a, 25-dihydroxyvitamin D3, 2a-methyl-19-nor-(20S)-1a,25-dihydroxyvitamin D3 (2AMD). Pancreatic tissues from control and 2AMD-treated Ins2-/-NOD mice were obtained weekly from 5 to 16 weeks of age. Using immunohistochemical (IHC) analysis, samples were analysed for changes in beta cell survival, islet structure and T cell invasion. Weekly intraperitoneal glucose tolerance tests (IPGTT) were performed to assess comparative beta cell function in control and treated animals. IHC demonstrated progressive beta cell destruction in control mice. In contrast, 2AMD treatment preserved islet cell architecture, arrested intra-islet T cell invasion and prevented the transition from insulitis to diabetes. IPGTT results revealed progressive impairment of beta cell function with increasing age in control mice, while 2AMD treatment resulted in normal beta function throughout the study. These results demonstrate that the Ins2 -/-NOD model provides a rapid and effective method for studying T1D and for assessing efficacy of anti-diabetic agents.