Interferon-α signaling promotes embryonic HSC maturation.

In the developing mouse embryo, the first hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) and mature as they transit through the fetal liver (FL). Compared with FL and adult HSCs, AGM HSCs have reduced repopulation potential in irradiated adult transplant recipients but mechanisms underlying this deficiency in AGM HSCs are poorly understood. By co-expression gene network analysis, we deduced that AGM HSCs show lower levels of interferon-α (IFN-α)/Jak-Stat1-associated gene expression than FL HSCs. Treatment of AGM HSCs with IFN-α enhanced long-term hematopoietic engraftment and donor chimerism. Conversely, IFN-α receptor-deficient AGMs (Ifnαr1(-/-)), had significantly reduced donor chimerism. We identify adenine-thymine-rich interactive domain-3a (Arid3a), a factor essential for FL and B lymphopoiesis, as a key transcriptional co-regulator of IFN-α/Stat1 signaling. Arid3a occupies the genomic loci of Stat1 as well as several IFN-α effector genes, acting to regulate their expression. Accordingly, Arid3a(-/-) AGM HSCs had significantly reduced transplant potential, which was rescued by IFN-α treatment. Our results implicate the inflammatory IFN-α/Jak-Stat pathway in the developmental maturation of embryonic HSCs, whose manipulation may lead to increased potency of reprogrammed HSCs for transplantation.

Smyd1 facilitates heart development by antagonizing oxidative and ER stress responses.

Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense-a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.

C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis.

The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3's oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors.

Structural and functional profiling of the human histone methyltransferase SMYD3.

The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the "split-SET" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.

The ARID family transcription factor bright is required for both hematopoietic stem cell and B lineage development.

Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice, its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that >99% of Bright(-/-) embryos die at midgestation from failed hematopoiesis. Bright(-/-) embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright(-/-) mice is markedly reduced. Rare survivors of lethality, which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b, suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody, B-1 responses to phosphocholine, and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation.

The L2a element is a mouse CD8 silencer that interacts with MAR-binding proteins SATB1 and CDP.

Previous transgenic-reporter and targeted-deletion studies indicate that the subset-specific expression of CD8αß heterodimers is controlled by multiple enhancer activities, since no silencer elements had been found within the locus. We have identified such a silencer as L2a, a previously characterized ∼ 220 bp nuclear matrix associating region (MAR) located ∼ 4.5 kb upstream of CD8α. L2a transgenes driven by the E8(I) enhancer showed no reporter expression in thymic subsets or T cells in splenic, inguinal and mesenteric lymph node peripheral T cells. Deletion of L2a resulted in significant reporter de-repression, even in the CD4(+)CD8(+) double positive (DP) thymocyte population. L2a contains binding sites for two MAR-interacting proteins, SATB1 and CDP. We found that that binding of these factors was markedly influenced by the content and spacing of L2a sub-motifs (L and S) and that SATB1 binds preferentially to the L motif both in vitro and in vivo. A small fraction of the transgenic CD8 single positive (SP) thymocytes and peripheral CD8(+) T cells bypassed L2a-silencing to give rise to variegated expression of the transgenic reporter. Crossing the L2a-containing transgene onto a SATB1 knockdown background enhanced variegated expression, suggesting that SATB1 is critical in overcoming L2a-silenced transcription.

Cardiac deletion of Smyd2 is dispensable for mouse heart development.

Chromatin modifying enzymes play a critical role in cardiac differentiation. Previously, it has been shown that the targeted deletion of the histone methyltransferase, Smyd1, the founding member of the SET and MYND domain containing (Smyd) family, interferes with cardiomyocyte maturation and proper formation of the right heart ventricle. The highly related paralogue, Smyd2 is a histone 3 lysine 4- and lysine 36-specific methyltransferase expressed in heart and brain. Here, we report that Smyd2 is differentially expressed during cardiac development with highest expression in the neonatal heart. To elucidate the functional role of Smyd2 in the heart, we generated conditional knockout (cKO) mice harboring a cardiomyocyte-specific deletion of Smyd2 and performed histological, functional and molecular analyses. Unexpectedly, cardiac deletion of Smyd2 was dispensable for proper morphological and functional development of the murine heart and had no effect on global histone 3 lysine 4 or 36 methylation. However, we provide evidence for a potential role of Smyd2 in the transcriptional regulation of genes associated with translation and reveal that Smyd2, similar to Smyd3, interacts with RNA Polymerase II as well as to the RNA helicase, HELZ.

Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development.

Proper thymocyte development is required to establish T-cell central tolerance and to generate naive T cells, both of which are essential for T-cell homeostasis and a functional immune system. Here we demonstrate that the loss of transcription factor Foxp1 results in the abnormal development of T cells. Instead of generating naive T cells, Foxp1-deficient single-positive thymocytes acquire an activated phenotype prematurely in the thymus and lead to the generation of peripheral CD4(+) T and CD8(+) T cells that exhibit an activated phenotype and increased apoptosis and readily produce cytokines upon T-cell receptor engagement. These results identify Foxp1 as an essential transcriptional regulator for thymocyte development and the generation of quiescent naive T cells.

m-Bop, a repressor protein essential for cardiogenesis, interacts with skNAC, a heart- and muscle-specific transcription factor.

The m-Bop protein encoded by the mouse Bop gene is strongly expressed in heart and skeletal muscle, and recent studies with Bop knockout mice have demonstrated that m-Bop is essential for cardiogenesis in vivo and can act as a HDAC-dependent repressor in vitro. In the present studies, m-Bop was observed to interact with skNAC, a reported transcriptional activator specific to heart and skeletal muscle. The amino-terminal S region of the split S-ET domain of m-Bop as well as the MYND domain were required for interaction with skNAC in both the two-hybrid system and in coimmunoprecipitation experiments from cultured mammalian cells. As shown previously for interaction of the MYND domain-containing transcriptional corepressor, BS69, with several viral and cellular oncoproteins, a PXLXP motif in skNAC was required for interaction with m-Bop. Similar kinetics of induction and localization of m-Bop and skNAC during the induction of myogenesis in cultured C2C12 cells suggests a possible associated role for these proteins during this process.

Bop encodes a muscle-restricted protein containing MYND and SET domains and is essential for cardiac differentiation and morphogenesis.

Many transcription factors regulate specific temporal-spatial events during cardiac differentiation; however, the mechanisms that regulate such events are largely unknown. Using a modified subtractive hybridization method to identify specific genes that influence early cardiac development, we found that Bop is expressed specifically in cardiac and skeletal muscle precursors before differentiation of these lineages. Bop encodes a protein containing MYND and SET domains, which have been shown to regulate transcription by mediating distinct chromatin modifications. We show that m-Bop is a histone deacetylase-dependent transcriptional repressor. Targeted deletion of Bop in mice disrupted maturation of ventricular cardiomyocytes and interfered with formation of the right ventricle. Normal expression of Hand2, a transcription factor essential for right ventricular development, in cardiomyocyte precursors is dependent upon m-Bop. These results indicate that m-Bop is essential for cardiomyocyte differentiation and cardiac morphogenesis.