Asifa Akhtar

The nuclear envelope and transcriptional control

Cells have evolved sophisticated multi-protein complexes that can regulate gene activity at various steps of the transcription process. Recent advances highlight the role of nuclear positioning in the control of gene expression and have put nuclear envelope components at centre stage. On the inner face of the nuclear envelope, active genes localize to nuclear-pore structures whereas silent chromatin localizes to non-pore sites. Nuclear-pore components seem to not only recruit the RNA-processing and RNA-export machinery, but contribute a level of regulation that might enhance gene expression in a heritable manner.

hMOF Histone Acetyltransferase Is Required for Histone H4 Lysine 16 Acetylation in Mammalian Cells

ABSTRACT Reversible histone acetylation plays an important role in regulation of chromatin structure and function. Here, we report that the human orthologue of Drosophila melanogaster MOF, hMOF, is a histone H4 lysine K16-specific acetyltransferase. hMOF is also required for this modification in mammalian cells. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by Western blot analysis and mass spectrometric analysis of endogenous histones. We also provide evidence that, similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 small interfering RNA-treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in the G2 and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by a reduction in hMOF protein levels. Furthermore, hMOF-depleted cells show an increased number of phospho-ATM and γH2AX foci and have an impaired repair response to ionizing radiation. Taken together, our data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression.

Nuclear Pore Proteins Nup153 and Megator Define Transcriptionally Active Regions in the Drosophila Genome

Transcriptional regulation is one of the most important processes for modulating gene expression. Though much of this control is attributed to transcription factors, histones, and associated enzymes, it is increasingly apparent that the spatial organization of chromosomes within the nucleus has a profound effect on transcriptional activity. Studies in yeast indicate that the nuclear pore complex might promote transcription by recruiting chromatin to the nuclear periphery. In higher eukaryotes, however, it is not known whether such regulation has global significance. Here we establish nucleoporins as a major class of global regulators for gene expression in Drosophila melanogaster. Using chromatin-immunoprecipitation combined with microarray hybridisation, we show that Nup153 and Megator (Mtor) bind to 25% of the genome in continuous domains extending 10 kb to 500 kb. These Nucleoporin-Associated Regions (NARs) are dominated by markers for active transcription, including high RNA polymerase II occupancy and histone H4K16 acetylation. RNAi–mediated knock-down of Nup153 alters the expression of ∼5,700 genes, with a pronounced down-regulatory effect within NARs. We find that nucleoporins play a central role in coordinating dosage compensation—an organism-wide process involving the doubling of expression of the male X chromosome. NARs are enriched on the male X chromosome and occupy 75% of this chromosome. Furthermore, Nup153-depletion abolishes the normal function of the male-specific dosage compensation complex. Finally, by extensive 3D imaging, we demonstrate that NARs contribute to gene expression control irrespective of their sub-nuclear localization. Therefore, we suggest that NAR–binding is used for chromosomal organization that enables gene expression control.

Dosage compensation in Drosophila melanogaster: epigenetic fine-tuning of chromosome-wide transcription.

Dosage compensation is an epigenetic mechanism that normalizes gene expression from unequal copy numbers of sex chromosomes. Different organisms have evolved alternative molecular solutions to this task. In Drosophila melanogaster, transcription of the single male X chromosome is upregulated by twofold in a process orchestrated by the dosage compensation complex. Despite this conceptual simplicity, dosage compensation involves multiple coordinated steps to recognize and activate the entire X chromosome. We are only beginning to understand the intriguing interplay between multiple levels of local and long-range chromatin regulation required for the fine-tuned transcriptional activation of a heterogeneous gene population. This Review highlights the known facts and open questions of dosage compensation in D. melanogaster.

Considerations when investigating lncRNA function in vivo

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome. DOI: http://dx.doi.org/10.7554/eLife.03058.001

The dMi-2 chromodomains are DNA binding modules important for ATP-dependent nucleosome mobilization

Drosophila Mi‐2 (dMi‐2) is the ATPase subunit of a complex combining ATP‐dependent nucleosome remodelling and histone deacetylase activities. dMi‐2 contains an HMG box‐like region, two PHD fingers, two chromodomains and a SNF2‐type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi‐2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi‐2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome‐stimulated ATPase activity. In contrast to dHP1, dMi‐2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi‐2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein–DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain‐associated activity.

The histone acetyltransferase hMOF is frequently downregulated in primary breast carcinoma and medulloblastoma and constitutes a biomarker for clinical outcome in medulloblastoma

Loss of H4 lysine 16 (H4K16) acetylation was shown to be a common feature in human cancer. However, it remained unclear which enzyme is responsible for the loss of this modification. Having recently identified the histone acetyltransferase human MOF (hMOF) to be required for bulk H4K16 acetylation, here we examined the involvement of hMOF expression and H4K16 acetylation in breast cancer and medulloblastoma. Analysis of a recent mRNA expression profiling study in breast cancer (n = 100 cases) and an array‐CGH screen in medulloblastomas (n = 102 cases), revealed downregulation in 40% and genomic loss in 11% of cases, respectively. We investigated hMOF protein expression as well as H4K16 acetylation in large series of primary breast carcinomas (n = 298) and primary medulloblastomas (n = 180) by immunohistochemistry. In contrast to nontransformed control tissues, significant fractions of both primary breast carcinomas and medulloblastomas showed markedly reduced hMOF mRNA and protein expression. In addition, hMOF protein expression tightly correlated with acetylation of H4K16 in all tested samples. For medulloblastoma, downregulation of hMOF protein expression was associated with lower survival rates identifying hMOF as an independent prognostic marker for clinical outcome in univariate as well as multivariate analyses.

Functional integration of the histone acetyltransferase MOF into the dosage compensation complex

Dosage compensation in flies involves doubling the transcription of genes on the single male X chromosome to match the combined expression level of the two female X chromosomes. Crucial for this activation is the acetylation of histone H4 by the histone acetyltransferase (HAT) MOF. In male cells, MOF resides in a complex (dosage compensation complex, DCC) with MSL proteins and noncoding roX RNA. Previous studies suggested that MOFs localization to the X chromosome was largely RNA‐mediated. We now found that contact of the MOF chromo‐related domain with roX RNA plays only a minor role in correct targeting to the X chromosome in vivo. Instead, a strong, direct interaction between a conserved MSL1 domain and a zinc finger within MOFs HAT domain is crucial. The functional consequences of this interaction were studied in vitro. Simultaneous contact of MOF with MSL1 and MSL3 led to its recruitment to chromatin, a dramatic stimulation of HAT activity and to improved substrate specificity. Activation of MOFs HAT activity upon integration into the DCC may serve to restrict the critical histone modification to the male X chromosome.

The Nonspecific Lethal Complex Is a Transcriptional Regulator in Drosophila

Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila.

Males absent on the first (MOF): from flies to humans.

Histone modifications such as acetylation, methylation and phosphorylation have been implicated in fundamental cellular processes such as epigenetic regulation of gene expression, organization of chromatin structure, chromosome segregation, DNA replication and DNA repair. Males absent on the first (MOF) is responsible for acetylating histone H4 at lysine 16 (H4K16) and is a key component of the MSL complex required for dosage compensation in Drosophila. The human ortholog of MOF (hMOF) has the same substrate specificity and recent purification of the human and Drosophila MOF complexes showed that these complexes were also highly conserved through evolution. Several studies have shown that loss of hMOF in mammalian cells leads to a number of different phenotypes; a G2/M cell cycle arrest, nuclear morphological defects, spontaneous chromosomal aberrations, reduced transcription of certain genes and an impaired DNA repair response upon ionizing irradiation. Moreover, hMOF is involved in ATM activation in response to DNA damage and acetylation of p53 by hMOF influences the cells decision to undergo apoptosis instead of a cell cycle arrest. These data, highlighting hMOF as an important component of many cellular processes, as well as links between hMOF and cancer will be discussed.