Kirby D. Johnson

Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic

The TGF-β (transforming growth factor-β)-related signalling proteins, including Decapentaplegic (Dpp) in Drosophila and bone morphogenic proteins and activin in vertebrates, affect the growth and patterning of a great variety of structures. However, the mechanisms by which these ligands regulate gene expression are not understood. Activation of complexes of type I with type II receptors results in the phosphorylation and nuclear localization of members of the SMAD protein family, which are thought to act as co-activators of transcription, perhaps in conjunction with sequence-specific cofactors. Here we show that the amino-terminal domain of the Drosophila Mothers against dpp protein (Mad), a mediator of Dpp signalling, possesses a sequence-specific DNA-binding activity that becomes apparent when carboxy-terminal residues are removed. Mad binds to and is required for the activation of an enhancer within the vestigial wing-patterning gene in cells across the entire developing wing blade. Mad also binds to Dpp-response elements in other genes. These results suggest that Dpp signalling regulates gene expression by activating Mad binding to target gene enhancers.

GATA Switches as Developmental Drivers

Transcriptional networks orchestrate complex developmental processes. Such networks are commonly instigated by master regulators of development. Considerable progress has been made in elucidating GATA factor-dependent genetic networks that control blood cell development. GATA-2 is required for the genesis and/or function of hematopoietic stem cells, whereas GATA-1 drives the differentiation of hematopoietic progenitors into a subset of the blood cell lineages. GATA-1 directly represses Gata2 transcription, and this involves GATA-1-mediated displacement of GATA-2 from chromatin, a process termed a GATA switch. GATA switches occur at numerous loci with critical functions, indicating that they are widely utilized developmental control tools.

Distinct Mechanisms Control RNA Polymerase II Recruitment to a Tissue-Specific Locus Control Region and a Downstream Promoter

Histone acetylation precedes activation of many genes. However, the establishment and consequences of long-range acetylation patterns are poorly understood. To define molecular determinants of the developmentally dynamic histone acetylation pattern of the beta-globin locus, we compared acetylation of the locus in MEL and CB3 erythroleukemia cells. CB3 cells lack the beta-globin locus control region (LCR) binding protein p45/NF-E2. We found that p45/NF-E2 was required for histone hyperacetylation at adult beta-globin promoters approximately 50 kilobases downstream of the LCR, but not at the LCR. Surprisingly, RNA polymerase II associated with the LCR in a p45/NF-E2-independent manner, while its recruitment to the promoter required p45/NF-E2. We propose that polymerase accesses the LCR and p45/NF-E2 induces long-range transfer of polymerase to the promoter, resulting in transcriptional activation.

Cooperative activities of hematopoietic regulators recruit RNA polymerase II to a tissue-specific chromatin domain

The hematopoietic transcription factor GATA-1 regulates erythropoiesis and β-globin expression. Although consensus GATA-1 binding sites exist throughout the murine β-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the β-globin promoters. The hematopoietic factor NF-E2 cooperated with GATA-1 to recruit pol II to the promoters. We propose that only when GATA-1 attracts pol II to the locus control region can pol II access the promoter in a NF-E2-dependent manner.

Dissecting long-range transcriptional mechanisms by chromatin immunoprecipitation.

Analysis of physiological mechanisms that control transcription often requires extrapolation of in vitro measurements into in vivo mechanisms. This extrapolation is complex, as mammalian genes are commonly organized into broad chromosomal domains, and such domains cannot be readily reconstituted in vitro. Thus, the nucleoprotein structure of chromosomes constitutes a considerable impediment to elucidating transcriptional mechanisms. The development of assays to measure protein-DNA interactions and chromatin structure in living cells has greatly facilitated progress in understanding physiological transcriptional mechanisms. Chromatin immunoprecipitation (ChIP) is a powerful approach that allows one to define the interaction of factors with specific chromosomal sites in living cells, thereby providing a snapshot of the native chromatin structure and factors bound to genes in different functional states. ChIP involves treating cells or tissue briefly with formaldehyde to crosslink proteins to DNA. An antibody against a protein suspected of binding a given cis-element is then used to immunoprecipitate chromatin fragments. Polymerase chain reaction analysis of the immunoprecipitate with primers flanking the cis-element reveals whether a specific DNA sequence is recovered in an immune-specific manner and therefore whether the protein contacted the site in living cells. The central focus of this review is the use of ChIP to study transcriptional activation over long distances on chromosomes.

Master regulatory GATA transcription factors: mechanistic principles and emerging links to hematologic malignancies

Numerous examples exist of how disrupting the actions of physiological regulators of blood cell development yields hematologic malignancies. The master regulator of hematopoietic stem/progenitor cells GATA-2 was cloned almost 20 years ago, and elegant genetic analyses demonstrated its essential function to promote hematopoiesis. While certain GATA-2 target genes are implicated in leukemogenesis, only recently have definitive insights emerged linking GATA-2 to human hematologic pathophysiologies. These pathophysiologies include myelodysplastic syndrome, acute myeloid leukemia and an immunodeficiency syndrome with complex phenotypes including leukemia. As GATA-2 has a pivotal role in the etiology of human cancer, it is instructive to consider mechanisms underlying normal GATA factor function/regulation and how dissecting such mechanisms may reveal unique opportunities for thwarting GATA-2-dependent processes in a therapeutic context. This article highlights GATA factor mechanistic principles, with a heavy emphasis on GATA-1 and GATA-2 functions in the hematopoietic system, and new links between GATA-2 dysregulation and human pathophysiologies.

GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome

Previous reports of GATA2 mutations have focused on the coding region of the gene or full gene deletions. We recently identified 2 patients with novel insertion/deletion mutations predicted to result in mRNA nonsense-mediated decay, suggesting haploinsufficiency as the mechanism of GATA2 deficient disease. We therefore screened patients without identified exonic lesions for mutations within conserved noncoding and intronic regions. We discovered 1 patient with an intronic deletion mutation, 4 patients with point mutations within a conserved intronic element, and 3 patients with reduced or absent transcription from 1 allele. All mutations affected GATA2 transcription. Full-length cDNA analysis provided evidence for decreased expression of the mutant alleles. The intronic deletion and point mutations considerably reduced the enhancer activity of the intron 5 enhancer. Analysis of 512 immune system genes revealed similar expression profiles in all clinically affected patients and reduced GATA2 transcript levels. These mutations strongly support the haploinsufficient nature of GATA2 deficiency and identify transcriptional mechanisms and targets that lead to MonoMAC syndrome.

Developmental control via GATA factor interplay at chromatin domains

Despite the extraordinary task of packaging mammalian DNA within the constraints of a cell nucleus, individual genes assemble into cell type‐specific chromatin structures with high fidelity. This chromatin architecture is a crucial determinant of gene expression signatures that distinguish specific cell types. Whereas extensive progress has been made on defining biochemical and molecular mechanisms of chromatin modification and remodeling, many questions remain unanswered about how cell type‐specific chromatin domains assemble and are regulated. This mini‐review will discuss emerging studies on how interplay among members of the GATA family of transcription factors establishes and regulates chromatin domains. Dissecting mechanisms underlying the function of hematopoietic GATA factors has revealed fundamental insights into the control of blood cell development from hematopoietic stem cells and the etiology of pathological states in which hematopoiesis is perturbed.

Hematopoietic-specific activators establish an overlapping pattern of histone acetylation and methylation within a mammalian chromatin domain

Posttranslational modification of histones through acetylation, methylation, and phosphorylation is a common mode of regulating chromatin structure and, therefore, diverse nuclear processes. One such modification, methylated histone H3 at lysine-4 (H3-meK4), colocalizes with hyperacetylated histones H3 and H4 in mammalian chromatin. Whereas activators directly recruit acetyltransferases, the process whereby H3-meK4 is established is unknown. We tested whether the hematopoietic-specific activators NF-E2 and GATA-1, which mediate transactivation of the β-globin genes, induce both histone acetylation and H3-meK4. Through the use of NF-E2- and GATA-1-null cell lines, we show that both activators induce H3 acetylation at the promoter upon transcriptional activation. However, analysis of H3-mek4 revealed that NF-E2 and GATA-1 differentially regulate chromatin modifications at the βmajor promoter. NF-E2, but not GATA-1, induces H3-meK4 at the promoter. Thus, under conditions in which NF-E2 and GATA-1 activate the transcription of an endogenous gene at least 570-fold, these activators differ in their capacity to induce H3-meK4. Despite strong H3-meK4 at hypersensitive site 2 of the upstream locus control region, neither factor was required to establish H3-meK4 at this site. These results support a model in which multiple tissue-specific activators collectively function to assemble a composite histone modification pattern, consisting of overlapping histone acetylation and methylation. As GATA-1 induced H3 acetylation, but not H3-meK4, at the promoter, H3 acetylation and H3-meK4 components of a composite histone modification pattern can be established independently.

Measurement of Protein-DNA Interactions In Vivo by Chromatin Immunoprecipitation

Elucidating mechanisms controlling nuclear processes requires an understanding of the nucleoprotein structure of genes at endogenous chromosomal loci. Traditional approaches to measuring protein-DNA interactions in vitro have often failed to provide insights into physiological mechanisms. Given that most transcription factors interact with simple DNA sequence motifs, which are abundantly distributed throughout a genome, it is essential to pinpoint the small subset of sites bound by factors in vivo. Signaling mechanisms induce the assembly and modulation of complex patterns of histone acetylation, methylation, phosphorylation, and ubiquitination, which are crucial determinants of chromatin accessibility. These seemingly complex issues can be directly addressed by a powerful methodology termed the chromatin immunoprecipitation (ChIP) assay. ChIP analysis involves covalently trapping endogenous proteins at chromatin sites, thereby yielding snapshots of protein-DNA interactions and histone modifications within living cells. The chromatin is sonicated to generate small fragments, and an immunoprecipitation is conducted with an antibody against the desired factor or histone modification. Crosslinks are reversed, and polymerase chain reaction (PCR) is used to assess whether DNA sequences are recovered immune-specifically. Chromatin-domain scanning coupled with quantitative analysis is a powerful means of dissecting mechanisms by which signaling pathways target genes within a complex genome.