👤 Brandon A Davies

AF4 gene, frequently translocated with mixed-lineage leukemia (MLL) in childhood acute leukemia, encodes a putative transcriptional activator of the AF4/LAF4/FMR2 (ALF) protein family previously implicated in lymphopoiesis and Purkinje cell function in the cerebellum. Here, we provide the first evidence for a direct role of AF4 in the regulation of transcriptional elongation by RNA polymerase II (Pol II). We demonstrate that mouse Af4 functions as a positive regulator of Pol II transcription elongation factor b (P-TEFb) kinase and, in complex with MLL fusion partners Af9, Enl and Af10, as a mediator of histone H3-K79 methylation by recruiting Dot1 to elongating Pol II. These pathways are interconnected and tightly regulated by the P-TEFb-dependent phosphorylation of Af4, Af9 and Enl which controls their transactivation activity and/or protein stability. Consistently, increased levels of phosphorylated Pol II and methylated H3-K79 are observed in the ataxic mouse mutant robotic, an over-expression model of Af4. Finally, we confirm the functional relevance of Af4, Enl and Af9 to the regulation of gene transcription as their over-expression strongly stimulates P-TEFb-dependent transcription of a luciferase reporter gene. Our findings uncover a central role for these proteins in the regulation of transcriptional elongation and coordinated histone methylation, providing valuable insight into their contribution to leukemogenesis and neurodegeneration. Since these activities likely extend to the entire ALF protein family, this study also significantly inputs our understanding of the molecular basis of FRAXE mental retardation syndrome in which FMR2 expression is silenced. Show less

In a screen designed to isolate Saccharomyces cerevisiae strains defective for in vitro chromatin assembly, two temperature-sensitive (ts) mutants were obtained: rmc1 and rmc3 (remodeling of chromatin). Cloning of RMC1 and RMC3 revealed a broad role for the ubiquitin-dependent targeting cascade as the ubiquitin-protein ligases (E3s), the anaphase promoting complex (APC; RMC1 encodes APC5) and Rsp5p, respectively, were identified. Genetic studies linked the rmc1/apc5 chromatin assembly defect to APC function: rmc1/apc5 genetically interacted with apc9Delta, apc10Delta, and cdc26Delta mutants. Furthermore, phenotypes associated with the rmc1/apc5 allele were consistent with defects in chromatin metabolism and in APC function: (i) UV sensitivity, (ii) plasmid loss, (iii) accumulation of G2/M cells, and (iv) suppression of the ts defect by growth on glucose-free media and by expression of ubiquitin. On the other hand, the multifunctional E3, Rsp5p, was shown to be required for both in vitro and in vivo chromatin assembly, as well as for the proper transcriptional and translational control of at least histone H3. The finding that the distinctly different E3 enzymes, APC and Rsp5p, both play roles in regulating chromatin assembly highlight the depth of the regulatory networks at play. The significance of these findings will be discussed. Show less

The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKbeta, p46 SAPKgamma (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKbeta and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21(ras) GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKbeta activation by p21(rac) (G12V), ERK1 activated by p21(ras) (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21(ras) was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases. Show less