Merlin Crossley

FOG, a Multitype Zinc Finger Protein, Acts as a Cofactor for Transcription Factor GATA-1 in Erythroid and Megakaryocytic Differentiation

The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages. Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, we have identified a novel, multitype zinc finger protein, Friend of GATA-1 (FOG), which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells. Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation. These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors.

Modification with SUMO: A role in transcriptional regulation

Small ubiquitin‐related modifier (SUMO) is a protein moiety that is ligated to lysine residues in a variety of target proteins. The addition of SUMO can modulate the ability of proteins to interact with their partners, alter their patterns of subcellular localization and control their stability. It is clear that SUMO influences many different biological processes, but recent data suggest that it is particularly important in the regulation of transcription. Indeed, several transcription factors, such as Sp3, c‐Jun, c‐Myb and various nuclear receptors, have recently been shown to be subject to sumoylation and, although this modification can have a positive influence, a growing body of evidence highlights its role in the negative regulation of transcription. This review summarizes recent experiments focusing on sumoylation and transcriptional repression.

Cloning and characterization of mCtBP2, a co‐repressor that associates with basic Krüppel‐like factor and other mammalian transcriptional regulators

Basic Krüppel‐like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. We have studied the activity of BKLF and found that it is capable of repressing transcription, and have mapped its repression domain to the N‐terminus. We carried out a two‐hybrid screen against BKLF and isolated a novel clone encoding murine C‐terminal‐binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein which binds to a Pro‐X‐Asp‐Leu‐Ser motif in the C‐terminus of the adenoviral oncoprotein, E1a. We show that mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA‐binding domain, mCtBP2 functions as a potent repressor. Finally, we demonstrate that mCtBP2 also interacts with the mammalian transcripition factors Evi‐1, AREB6, ZEB and FOG. These results establish a new member of the CtBP family, mCtBP2, as a mammalian co‐repressor targeting diverse transcriptional regulators.

Mammalian Krüppel-like transcription factors: more than just a pretty finger

The transcription factor SP1 contains three Krüppel-like zinc fingers. Recently, several related proteins, including erythroid, lung and gut-enriched Krüppel-like factors, have been identified. Together with SP1, these proteins form a sizeable family of transcription factors that share homology in their zinc-finger domains but differ elsewhere. Analysis of these differences is illuminating specific mechanisms by which transcription is regulated.

Transcriptional cofactors of the FOG family interact with GATA proteins by means of multiple zinc fingers.

Friend of GATA‐1 (FOG‐1) is a zinc finger protein that has been shown to interact physically with the erythroid DNA‐binding protein GATA‐1 and modulate its transcriptional activity. Recently, two new members of the FOG family have been identified: a mammalian protein, FOG‐2, that also associates with GATA‐1 and other mammalian GATA factors; and U‐shaped, a Drosophila protein that interacts with the Drosophila GATA protein Pannier. FOG proteins contain multiple zinc fingers and it has been shown previously that the sixth finger of FOG‐1 interacts specifically with the N‐finger but not the C‐finger of GATA‐1. Here we show that fingers 1, 5 and 9 of FOG‐1 also interact with the N‐finger of GATA‐1 and that FOG‐2 and U‐shaped also contain multiple GATA‐interacting fingers. We define the key contact residues and show that these residues are highly conserved in GATA‐interacting fingers. We examine the effect of selectively mutating the four interacting fingers of FOG‐1 and show that each contributes to FOG‐1s ability to modulate GATA‐1 activity. Finally, we show that FOG‐1 can repress GATA‐1‐mediated activation and present evidence that this ability involves the recently described CtBP co‐repressor proteins that recognize all known FOG proteins.

ISOLATION AND CHARACTERIZATION OF THE CDNA ENCODING BKLF/TEF-2, A MAJOR CACCC-BOX-BINDING PROTEIN IN ERYTHROID CELLS AND SELECTED OTHER CELLS

CACCC boxes are among the critical sequences present in regulatory elements of genes expressed in erythroid cells, as well as in selected other cell types. While an erythroid cell-specific CACCC-box-binding protein, EKLF, has been shown to be required in vivo for proper expression of the adult beta-globin gene, it is dispensable for the regulation of several other globin and nonglobin erythroid cell-expressed genes. In the work described here, we searched for additional CACCC-box transcription factors that might be active in murine erythroid cells. We identified a major gel shift activity (termed BKLF), present in yolk sac and fetal liver erythroid cells, that could be distinguished from EKLF by specific antisera. Through relaxed-stringency hybridization, we obtained the cDNA encoding BKLF, a highly basic, novel zinc finger protein that is related to EKLF and other Krüppel-like members in its DNA-binding domain but unrelated elsewhere. BKLF, which is widely but not ubiquitously expressed in cell lines, is highly expressed in the midbrain region of embryonic mice and appears to correspond to the gel shift activity TEF-2, a transcriptional activator implicated in regulation of the simian virus 40 enhancer and other CACCC-box-containing regulatory elements. Because BKLF binds with high affinity and preferentially over Sp1 to many CACCC sequences of erythroid cell expressed genes, it is likely to participate in the control of many genes whose expression appears independent of the action of EKLF.

Self-Association of the Erythroid Transcription Factor GATA-1 Mediated by Its Zinc Finger Domains

GATA-1, the founding member of a distinctive family of transcription factors, is expressed predominantly in erythroid cells and participates in the expression of numerous erythroid cell-expressed genes. GATA-binding sites are found in the promoters and enhancers of globin and nonglobin erythroid genes as well as in the alpha- and beta-globin locus control regions. To elucidate how GATA-1 may function in a variety of regulatory contexts, we have examined its protein-protein interactions. Here we show that GATA-1 self-associates in solution and in whole-cell extracts and that the zinc finger region of the molecule is sufficient to mediate this interaction. This physical interaction can influence transcription, as GATA-1 self-association is able to recruit a transcriptionally active but DNA-binding-defective derivative of GATA-1 to promoter-bound GATA-1 and result in superactivation. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, we have localized the minimal domain required for GATA-1 self-association to 40 amino acid residues within the C-terminal zinc finger region. Finally, we have detected physical interaction of GATA-1 with other GATA family members (GATA-2 and GATA-3) also mediated through the zinc finger domain. These findings have broad implications for the involvement of GATA factors in transcriptional control. In particular, the interaction of GATA-1 with itself and with other transcription factors may facilitate its function at diverse promoters in erythroid cells and also serve to bring together, or stabilize, loops between distant regulatory elements, such as the globin locus control regions and downstream globin promoters. We suggest that the zinc finger region of GATA-1, and related proteins, is multifunctional and mediates not only DNA binding but also important protein-protein interactions.

The C-terminal zinc finger of GATA-1 or GATA-2 is sufficient to induce megakaryocytic differentiation of an early myeloid cell line.

The GATA-1 and GATA-2 transcription factors, which each contain two homologous zinc fingers, are important hematopoietic regulators expressed within the erythroid, mast cell, and megakaryocytic lineages. Enforced expression of either factor in the primitive myeloid line 416B induces megakaryocytic differentiation. The features of their structure required for this activity have been explored. The ability of 12 GATA-1 mutants to promote 416B maturation was compared with their DNA-binding activity and transactivation potential. Differentiation did not require any of the seven serine residues that are phosphorylated in vivo, an N-terminal region bearing the major transactivation domain, or a C-terminal segment beyond the fingers. Removal of a consensus nuclear localization signal following the second finger did not block differentiation or nuclear translocation. The N-terminal finger was also dispensable, although its removal attenuated differentiation. In contrast, the C-terminal finger was essential, underscoring its distinct function. Remarkably, only 69 residues spanning the C-terminal finger were required to induce limited megakaryocytic differentiation. Analysis of three GATA-2 mutants led to the same conclusion. Endogenous GATA-1 mRNA was induced by most mutants and may contribute to differentiation. Because the GATA-1 C-terminal finger could bind its target site but not transactivate a minimal reporter, it may direct megakaryocytic maturation by derepressing specific genes and/or by interacting with another protein which provides the transactivation function.

Controlling the size of lipid droplets: lipid and protein factors

Recent advances have transformed our understanding of lipid droplets (LDs). Once regarded as inert lipid storage granules, LDs are now recognized as multi-functional organelles that affect many aspects of cell biology and metabolism. However, fundamental questions concerning the biogenesis and growth of LDs remain unanswered. Recent studies have uncovered novel modes of LD growth (including rapid/homotypic as well as slow/atypical LD fusion), and identified key proteins (e.g. Fsp27, seipin, FITM2 and perilipin 1) and lipids (e.g. phosphatidylcholine and phosphatidic acid) that regulate the size of LDs. Phospholipids appear to have an evolutionarily conserved role in LD growth. Protein factors may regulate LD expansion directly and/or indirectly through modulating the level and composition of phospholipids on LD surface.

Protein-Protein Interaction between Fli-1 and GATA-1 Mediates Synergistic Expression of Megakaryocyte-Specific Genes through Cooperative DNA Binding

ABSTRACT Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each others activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbα in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIbα genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.