Chhaya Das

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.

Functional studies of BCL11A: characterization of the conserved BCL11A-XL splice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells

BackgroundChromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development.ResultsAlternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles.ConclusionWe propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.

Regulation of matrix attachment region-dependent, lymphocyte-restricted transcription through differential localization within promyelocytic leukemia nuclear bodies.

Bright (B cell regulator of IgH transcription) transactivates the immunoglobulin heavy chain (IgH) intronic enhancer, Eμ, by binding to matrix attachment regions (MARs), sites necessary for DNA attachment to the nuclear matrix. Here we report that Bright interacts with the ubiquitous autoantigen Sp100, a component of promyelocytic leukemia nuclear bodies (PML NBs), and with LYSp100B/Sp140, the lymphoid‐restricted homolog of Sp100. Both in intact cells and in nuclear matrix preparations, the majority of Bright and Sp100 colocalize within PML NBs. In contrast, Bright colocalizes with only a small fraction of LYSp100B while inducing a redistribution of the majority of LYSp100B from its associated nuclear domains (LANDs) into nucleoplasm and cytoplasm. Sp100 represses the MAR‐binding and transactivation activity of Bright. LYSp100B interacts more weakly with Bright but requires significantly higher levels than Sp100 to inhibit MAR binding. However, it strongly stimulates Bright transactivation through Eμ. We suggest that Sp100 and LYSp100B interactions with Bright have different consequences for IgH transcription, potentially through differential association of Eμ MARs with nuclear matrix‐ associated PML NBs and LANDs.

REKLES is an ARID3-restricted multifunctional domain.

Bright/Dril1/ARID3a is a B cell-specific, matrix association (or attachment) region-binding transcriptional regulator of immunoglobulin heavy chain genes and of E2F1-dependent cell cycle progression. Bright contains a central DNA binding domain termed ARID (AT-rich interacting domain) and a C-terminal region termed REKLES (for a conserved amino acid motif). The ARID domain has been identified in seven highly conserved families of metazoan proteins (ARID1-5 and JARID1-2), whereas REKLES is found only in the ARID3 subfamily (composed of Bright/ARID3a, Bdp/ARID3b, and Bright-like/ARID3c). REKLES consists of two subdomains: a modestly conserved N-terminal REKLESα and a highly conserved (among ARID3 orthologous proteins) C-terminal REKLESβ. Previously we showed that Bright undergoes nucleocytoplasmic shuttling and that REKLESα and -β were required, respectively, for nuclear import and Crm1-dependent nuclear export. Here we show that Bright further requires REKLESβ for self-association or paralogue association and for nuclear matrix targeting. REK-LES promotes and regulates the extent of Bright multimerization, which occurs in the absence or presence of target DNA and is necessary for specific DNA binding. REKLESβ-mediated interaction of Bright with Bdp, which localizes strictly to the nucleus, traps Bright within the nucleus via neutralization of its nuclear export activity. These results identify REKLES as a multifunctional domain that has co-evolved with and regulates functional properties of the ARID3 DNA binding domain.

Arid3b Is Critical for B Lymphocyte Development.

Arid3a and Arid3b belong to a subfamily of ARID (AT-rich interaction domain) transcription factors. The Arid family is involved in regulating chromatin accessibility, proliferation, and differentiation. Arid3a and Arid3b are closely related and share a unique REKLES domain that mediates their homo- and hetero-multimerization. Arid3a was originally isolated as a B cell transcription factor binding to the AT rich matrix attachment regions (MARS) of the immunoglobulin heavy chain intronic enhancer. Deletion of Arid3a results in a highly penetrant embryonic lethality with severe defects in erythropoiesis and hematopoietic stem cells (HSCs). The few surviving Arid3a-/- (<1%) animals have decreased HSCs and early progenitors in the bone marrow, but all mature lineages are normally represented in the bone marrow and periphery except for B cells. Arid3b-/- animals die around E7.5 precluding examination of hematopoietic development. So it is unclear whether the phenotype of Arid3a loss on hematopoiesis is dependent or independent of Arid3b. In this study we circumvented this limitation by also examining hematopoiesis in mice with a conditional allele of Arid3b. Bone marrow lacking Arid3b shows decreased common lymphoid progenitors (CLPs) and downstream B cell populations while the T cell and myeloid lineages are unchanged, reminiscent of the adult hematopoietic defect in Arid3a mice. Unlike Arid3a-/- mice, HSC populations are unperturbed in Arid3b-/- mice. This study demonstrates that HSC development is independent of Arid3b, whereas B cell development requires both Arid3a and Arid3b transcription factors.

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