Numerous studies in multiple systems support that histone H3 lysine 36 di-methylation (H3K36me2) is associated with transcriptional activation, however the underlying mechanisms are not well defined. Here we show that the H3K36me2 chromatin mark written by the ASH1L histone methyltransferase is preferentially bound in vivo by LEDGF, an MLL-associated protein that co-localizes with MLL, ASH1L and H3K36me2 on chromatin genome wide. Furthermore, ASH1L facilitates recruitment of LEDGF and wild type MLL proteins to chromatin at key leukemia target genes, and is a crucial regulator of MLL-dependent transcription and leukemic transformation. Conversely KDM2A, an H3K36me2 demethylase and Polycomb-group silencing protein, antagonizes MLL-associated leukemogenesis. Our studies are the first to provide a basic mechanistic insight into epigenetic interactions wherein placement, interpretation and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a novel target for therapeutic intervention.

Five members of the KMT2 family of lysine methyltransferases, originally named the mixed lineage leukemia (MLL1-5) proteins, regulate gene expression during embryogenesis and development. Each KMT2A-E contains a catalytic SET domain that methylates lysine 4 of histone H3, and one or several PHD fingers. Over the past few years a growing number of studies have uncovered diverse biological roles of the KMT2A-E PHD fingers, implicating them in binding to methylated histones and other nuclear proteins, and in mediating the E3 ligase activity and dimerization. Mutations in the PHD fingers or deletion of these modules are linked to human diseases including cancer and Kabuki syndrome. In this work, we summarize recently identified biological functions of the KMT2A-E PHD fingers, discuss mechanisms of their action, and examine preference of these domains for histone and non-histone ligands.

Actaea racemosa L. (black cohosh; syn. Cimicifuga racemosa L. Nutt.) is a native North American perennial whose root and rhizome preparations are commercially available as phytomedicines and dietary supplements, primarily for management of menopausal symptoms. Despite its wide use, methods that accurately identify processed A. racemosa are not well established; product adulteration remains a concern. Because of its similar appearance and growing locales, A. racemosa has been unintentionally mixed with other species of the genus, such as Actaea pachypoda Ell. (white cohosh) and more commonly Actaea podocarpa DC. (yellow cohosh). The genus Actaea also has 23 temperate species with numerous common names, which can also contribute to the misidentification of plant material. Consequently, a variety of Actaea spp. are common adulterants of commercially available black cohosh preparations. Thin-layer chromatography (TLC) and combined TLC-bioluminescence (Bioluminex) are efficient, economical, and effective techniques which provide characteristic patterns and toxicity profiles for each plant species. These data indicate that common black cohosh adulterants, such as yellow cohosh, can be differentiated from black cohosh by TLC and TLC-bioluminescence. This study also showed that unknown contaminants that were not detected using standard A. racemosa identity techniques were readily detected by TLC and TLC-bioluminescence.

<p>Supplementary Figure 1 shows NMR and biochemical data in support of LEDGF nucleosome binding specificity. Supplementary Figure 2 shows chromatin IP data indicating requirement for LEDGF in MLL target gene localization. Supplementary Figure 3 shows global and gene-specific chromatin localization of ASH1L. Supplementary Figure 4 shows quantitation of differentiation and apoptosis following ASH1L knockdown. Supplementary Figure 5 shows that over-expression of KDM2A interferes with MLL-mediated leukemic transformation phenotypes.</p>

Hormone secretion form anterior pituitary cells is known to be regulated by G protein coupled receptors (GPCRs) that regulate intracellular cAMP. The objective of this study was to determine mechanisms by which elevated cAMP control intracellular calcium. We have used fura‐2 measurements of intracellular calcium concentration along with both population biochemical cAMP measurements and single cell FRET‐based cAMP measurements to investigate the relationship between [Ca2+] and cAMP in clonal rat MMQ lactotrophs. Activation vasoactive intestinal peptide receptors that are coupled to Gs led to Ca2+ influx as evident by increase in Ca2+‐oscillations. Bypass of GPCRs with the adenylyl cylclase activator forskolin also resulted in strong in increase in Ca2+ oscillations consistent with measured forskolin‐induced increase in [cAMP]. Pharmacological activation of PKA using 6‐Bn‐cAMP or activation of EPAC using 8‐cpt‐cAMP each resulted in increase in Ca2+ indicating a role for both cAMP binding proteins in control of calcium dynamics although with differing lag times likely reflecting distinct signaling pathways. The broad‐spectrum cAMP phosphodiesterase (PDE) inhibitor IBMX caused an increase in Ca2+ influx without delay. The PDE3 inhibitor milrinone caused a strong increase in Ca2+ after a delay whereas the PDE4 inhibitor rolipram caused an immediate increase in Ca2+. Roliparam also triggered apparent store release. FRET‐based measurements of [cAMP] are being used to further analyze these differences. These results suggest a possible different relationship between the various PDE sensitive pools of cAMP and Ca2+ ‐channels as well as distinct roles for PKA and EPAC in control of Ca2+ dynamics and secretion.