The Chromatin Remodeling Protein ATRX in Development and Maintenance of Mouse Skeletal Tissues

Alpha-thalassemia X-linked mental retardation (ATR-X) syndrome is a rare genetic disorder associated with severe developmental delay, mental retardation and craniofacial dysmorphism. This syndrome is caused by mutations in the ATRX gene which encodes a member of the SWI/SNF family of chromatin remodeling proteins. ATR-X patients exhibit dwarfism and skeletal defects, including hand and foot deformities. I hypothesized that the skeletal deformities in ATR-X syndrome are due to a direct role of ATRX in the development of the skeleton. My objective was to characterise skeletal phenotypes observed in three animal models conditionally deficient for ATRX in different skeletal tissues. Mice lacking the Atrx gene in forelimb mesenchyme, cartilage, or bone were generated using the Cre-lox system with Cre-recombinase under control of Prx1, Col2a1 or Col1a2 promoters, respectively. Mice lacking ATRX in cartilage or bone displayed limited skeletal phenotypes and did not recapitulate the defects seen in ATR-X syndrome. Loss of ATRX protein earlier during limb development lead to shortening of the distal phalanges which correlated with increased DNA damage and apoptotic cell death in the mesenchyme of developing digits. This phenotype resembles brachydactyly in human ATR-X syndrome patients and suggests that ATRX is involved in maintaining genome integrity in limb tissue. Aging mice lacking ATRX in skeletal tissues did not show any increased susceptibility to osteoarthritis. In summary, this study suggests that skeleton-intrinsic roles of ATRX are responsible for some but not all of the skeletal deformities observed in ATR-X syndrome

A role for ATP Citrate Lyase in cell cycle regulation during myeloid differentiation

Differentiation of myeloid progenitor cells into macrophages is accompanied by increased PU.1 concentration and increasing cell cycle length, culminating in cell cycle arrest. Induction of PU.1 expression in a cultured myeloid cell line expressing low PU.1 concentration results in decreased levels of mRNA encoding ATP-Citrate Lyase (ACL) and cell cycle arrest. ACL is an essential enzyme for generating acetyl-CoA, a key metabolite for the first step in fatty acid synthesis and for histone acetylation. We hypothesized that ACL may play a role in cell cycle regulation in the myeloid lineage. In this study, we found that acetyl-CoA or acetate supplementation was sufficient to rescue cell cycle progression in cultured BN cells treated with an ACL inhibitor or induced for PU.1 expression. Acetyl-CoA supplementation was also sufficient to rescue cell cycle progression in BN cells treated with a fatty acid synthase (FASN) inhibitor. We demonstrated that acetyl-CoA was utilized in both fatty acid synthesis and histone acetylation pathways to promote proliferation. Finally, we found that Acly mRNA transcript levels decrease during normal macrophage differentiation from bone marrow precursors. Our results suggest that regulation of ACL activity is a potentially important point of control for cell cycle regulation in the myeloid lineage

Loss of ATRX does not confer susceptibility to osteoarthritis

The chromatin remodelling protein ATRX is associated with the rare genetic disorder ATR-X syndrome. This syndrome includes developmental delay, cognitive impairment, and a variety of skeletal deformities. ATRX plays a role in several basic chromatin-mediated cellular events including DNA replication, telomere stability, gene transcription, and chromosome congression and cohesion during cell division. We have used a loss-of-function approach to directly investigate the role of Atrx in the adult skeleton in three different models of selective Atrx loss. We specifically targeted deletion of Atrx to the forelimb mesenchyme, to cartilage and to bone-forming osteoblasts. We previously demonstrated that loss of ATRX in forelimb mesenchyme causes brachydactyly while deletion in chondrocytes had minimal effects during development. We now show that targeted deletion of Atrx in osteoblasts causes minor dwarfism but does not recapitulate most of the skeletal phenotypes seen in ATR-X syndrome patients. In adult mice from all three models, we find that joints lacking Atrx are not more susceptible to osteoarthritis, as determined by OARSI scoring and immunohistochemistry. These results indicate that while ATRX plays limited roles during early stages of skeletal development, deficiency of the protein in adult tissues does not confer susceptibility to osteoarthritis. © 2013 Solomon et al

Loss of ATRX does not confer susceptibility to osteoarthritis

The chromatin remodelling protein ATRX is associated with the rare genetic disorder ATR-X syndrome. This syndrome includes developmental delay, cognitive impairment, and a variety of skeletal deformities. ATRX plays a role in several basic chromatin-mediated cellular events including DNA replication, telomere stability, gene transcription, and chromosome congression and cohesion during cell division. We have used a loss-of-function approach to directly investigate the role of Atrx in the adult skeleton in three different models of selective Atrx loss. We specifically targeted deletion of Atrx to the forelimb mesenchyme, to cartilage and to bone-forming osteoblasts. We previously demonstrated that loss of ATRX in forelimb mesenchyme causes brachydactyly while deletion in chondrocytes had minimal effects during development. We now show that targeted deletion of Atrx in osteoblasts causes minor dwarfism but does not recapitulate most of the skeletal phenotypes seen in ATR-X syndrome patients. In adult mice from all three models, we find that joints lacking Atrx are not more susceptible to osteoarthritis, as determined by OARSI scoring and immunohistochemistry. These results indicate that while ATRX plays limited roles during early stages of skeletal development, deficiency of the protein in adult tissues does not confer susceptibility to osteoarthritis. © 2013 Solomon et al

Targeted loss of the ATR-X syndrome protein in the limb mesenchyme of mice causes brachydactyly

ATR-X syndrome is a rare genetic disorder caused by mutations in the ATRX gene. Affected individuals are cognitively impaired and display a variety of developmental abnormalities, including skeletal deformities. To investigate the function of ATRX during skeletal development, we selectively deleted the gene in the developing forelimb mesenchyme of mice. The absence of ATRX in the limb mesenchyme resulted in shorter digits, or brachydactyly, a defect also observed in a subset of ATR-X patients. This phenotype persisted until adulthood, causing reduced grip strength and altered gait in mutant mice. Examination of the embryonic ATRX-null forelimbs revealed a significant increase in apoptotic cell death, which could explain the reduced digit length. In addition, staining for the DNA damage markers γ-histone 2A family member X (γ-H2AX) and 53BP1 demonstrated a significant increase in the number of cells with DNA damage in the embryonic ATRX-null forepaw. Strikingly, only one large bright DNA damage event was observed per nucleus in proliferating cells. These large γ-H2AX foci were located in close proximity to the nuclear lamina and remained largely unresolved after cell differentiation. In addition, ATRX-depleted forelimb mesenchymal cells did not exhibit hypersensitivity to DNA fork-stalling compounds, suggesting that the nature as well as the response to DNA damage incurred by loss of ATRX in the developing limb fundamentally differs from other tissues. Our data suggest that DNA damage-induced apoptosis is a novel cellular mechanism underlying brachydactyly that might be relevant to additional skeletal syndromes. © The Author 2013. Published by Oxford University Press. All rights reserved

Identification of a negative regulatory role for Spi-C in the murine B cell lineage

Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib-/-Spic+/-). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib-/- mouse spleens. Spib-/-Spic+/- B cells had restored proliferation compared with Spib-/- B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib-/-Spic+/- phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib-/-Spic+/- B cells compared with Spib-/- B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function

The effect of maternal protein restriction during perinatal life on the inflammatory response in pediatric rats

Fetal growth restriction can affect health outcomes in postnatal life. This study tested the hypothesis that the response to an inflammatory pulmonary insult is altered in pediatric fetal growth restricted rats. Using a low-protein diet during gestation and postnatal life, growth-restricted male and female rats and healthy control rats were exposed to an inflammatory insult via the intratracheal instillation of heat-killed bacteria. After 6 h, animal lungs were examined for lung inflammation and status of the surfactant system. The results showed that in response to an inflammatory insult, neutrophil infiltration was decreased in both male and female rats in the growth-restricted animals compared with the control rats. The amount of surfactant was increased in the growth-restricted animals compared with the control rats, regardless of the inflammatory insult. It is concluded that fetal growth restriction results in increased surfactant and altered neutrophil responses following pulmonary insult

PU.1 regulates Ig light chain transcription and rearrangement in pre-B cells during B cell development

B cell development and Ig rearrangement are governed by cell type- and developmental stage-specific transcription factors. PU.1 and Spi-B are E26-transformation-specific transcription factors that are critical for B cell differentiation. To determine whether PU.1 and Spi-B are required for B cell development in the bone marrow, Spi1 (encoding PU.1) was conditionally deleted in B cells by Cre recombinase under control of the Mb1 gene in Spib (encoding Spi-B)-deficient mice. Combined deletion of Spi1 and Spib resulted in a lack of mature B cells in the spleen and a block in B cell development in the bone marrow at the small pre-B cell stage. To determine target genes of PU.1 that could explain this block, we applied a gain-of-function approach using a PU.1/Spi-B- deficient pro-B cell line in which PU.1 can be induced by doxycycline. PU.1-induced genes were identified by integration of chromatin immunoprecipitation-sequencing and RNA-sequencing data. We found that PU.1 interacted with multiple sites in the Igk locus, including Vk promoters and regions located downstream of Vk second exons. Induction of PU.1 induced Igk transcription and rearrangement. Upregulation of Igk transcription was impaired in small pre-B cells from PU.1/Spi-B-deficient bone marrow. These studies reveal an important role for PU.1 in the regulation of Igk transcription and rearrangement and a requirement for PU.1 and Spi-B in B cell development

Nfkb1 activation by the E26 transformation-specific transcription factors PU.1 and Spi-B promotes toll-like receptor-mediated splenic B cell proliferation

Generation of antibodies against T-independent and T-dependent antigens requires Toll-like receptor (TLR) engagement on B cells for efficient responses. However, the regulation of TLR expression and responses in B cells is not well understood. PU.1 and Spi-B (encoded by Sfpi1 and Spib, respectively) are transcription factors of the E26 transformation-specific (ETS) family and are important for B cell development and function. It was found that B cells from mice knocked out for Spi-B and heterozygous for PU.1 (Sfpi1+/- Spib-/- [PUB] mice) proliferated poorly in response to TLR ligands compared to wild-type (WT) B cells. The NF-κB family member p50 (encoded by Nfkb1) is required for lipopolysaccharide (LPS) responsiveness in mice. PUB B cells expressed reduced Nfkb1 mRNA transcripts and p50 protein. The Nfkb1 promoter was regulated directly by PU.1 and Spi-B, as shown by reporter assays and chromatin immunoprecipitation analysis. Occupancy of the Nfkb1 promoter by PU.1 was reduced in PUB B cells compared to that in WT B cells. Finally, infection of PUB B cells with a retroviral vector encoding p50 substantially restored proliferation in response to LPS. We conclude that Nfkb1 transcriptional activation by PU.1 and Spi-B promotes TLR-mediated B cell proliferation

Genome-Wide Comparison of PU.1 and Spi-B Binding Sites in a Mouse B Lymphoma Cell Line

Background Spi-B and PU.1 are highly related members of the E26-transformation-specific (ETS) family of transcription factors that have similar, but not identical, roles in B cell development. PU.1 and Spi-B are both expressed in B cells, and have been demonstrated to redundantly activate transcription of genes required for B cell differentiation and function. It was hypothesized that Spi-B and PU.1 occupy a similar set of regions within the genome of a B lymphoma cell line. Results To compare binding regions of Spi-B and PU.1, murine WEHI-279 lymphoma cells were infected with retroviral vectors encoding 3XFLAG-tagged PU.1 or Spi-B. Anti-FLAG chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) was performed. Analysis for high-stringency enriched genomic regions demonstrated that PU.1 occupied 4528 regions and Spi-B occupied 3360 regions. The majority of regions occupied by Spi-B were also occupied by PU.1. Regions bound by Spi-B and PU.1 were frequently located immediately upstream of genes associated with immune response and activation of B cells. Motif-finding revealed that both transcription factors were predominantly located at the ETS core domain (GGAA), however, other unique motifs were identified when examining regions associated with only one of the two factors. Motifs associated with unique PU.1 binding included POU2F2, while unique motifs in the Spi-B regions contained a combined ETS-IRF motif. Conclusions Our results suggest that complementary biological functions of PU.1 and Spi-B may be explained by their interaction with a similar set of regions in the genome of B cells. However, sites uniquely occupied by PU.1 or Spi-B provide insight into their unique functions