serpent
Biological oscillations are observed at many levels of cellular organization. In the social amoeba Dictyostelium discoideum, starvation-triggered multicellular development is organized by periodic cyclic adenosine 3',5'-monophosphate (cAMP) waves, which provide both chemoattractant gradients and developmental signals. GtaC, a GATA transcription factor, was shown to exhibit rapid nucleocytoplasmic shuttling in response to cAMP waves. This behavior requires coordinated action of a nuclear localization signal and reversible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor-mediated phosphorylation. Although both are required for developmental gene expression, receptor occupancy promotes nuclear exit of GtaC, which leads to a transient burst of transcription at each cAMP cycle. This biological circuit filters out high-frequency signals and counts those admitted, thereby enabling cells to modulate gene expression according to the dynamic pattern of the external stimuli (Cai, 2014).
GATA transcription factors in C. elegans The endoderm in the nematode Caenorhabditis elegans is clonally derived from the E founder cell. A single genomic region (the endoderm-determining region, or EDR) is required for the production of the entire C. elegans endoderm. In embryos lacking the EDR, the E cell gives rise to ectoderm and mesoderm, instead of endoderm, and appears to adopt the fate of its cousin, the C
founder cell. end-1, a gene from the EDR, restores endoderm production in EDR deficient homozygotes. end-1 transcripts are first detectable specifically in the E cell, consistent with a direct role for end-1 in endoderm development. The END-1 protein is an apparent zinc finger-containing GATA transcription factor. Since GATA factors have been implicated in endoderm development in other animals, these findings suggest that endoderm may be specified by molecularly conserved mechanisms in triploblastic animals. It is proposed that end-1, the first zygotic gene known to be involved in the
specification of germ layer and founder cell identity in C. elegans, may link maternal genes that regulate the establishment of the endoderm to downstream genes responsible for endoderm differentiation (Zhu, 1997).
The END-1 GATA factor has been implicated in specifying endoderm in Caenorhabditis elegans. It is the earliest
known zygotic protein expressed in the lineage of E, the clonal endoderm progenitor. Ubiquitous end-1
expression during a critical period in embryogenesis causes all non-endodermal lineages to produce endoderm instead of ectoderm and/or mesoderm. To investigate further whether ectopic endoderm production might reflect transformations in early blastomere fates, an examination was carried out to see whether ectopic END-1 not only activates expression of endodermal
markers but also suppresses expression of markers characteristic of nonendodermal lineages. Expression of nonendodermal lineage markers from isolated blastomeres, including the
LIN-26 ectodermal marker from AB, body muscle from P2, and pharynx muscle from MS, is attenuated in a heat shock-dependent manner in hs-end-1 embryos, concomitant with ectopic production of intestine. Thus, activation of endoderm development by END-1 apparently results in coincident repression of genetic programs that promote nonendodermal cell types. The optimal period for response (~20-50 cells)
coincides with the time at which end-1 is normally expressed in wild-type embryos. Nonetheless, end-1 can cause a transformation in cell fate well beyond the stage at which the
founder cell lineages are established and differentiation potential is normally restricted to particular
blastomeres. In embryos heat-shocked after ~200 cells, although expression from the heat shock promoter is still robust, morphological characteristics and molecular markers of normal differentiation from all germ
layers are seen, suggesting that most cells had become committed to their normal differentiated fates
and are not capable of being reprogrammed by end-1. Thus, competency for reprogramming to endoderm occurs during a critical period in development. END-1 expression bypasses the requirement for maternal SKN-1 (a novel protein) and the maternal Wnt signaling pathway in endoderm formation. SKN-1 binds to end-1 regulatory sequences through SKN-1 consensus binding sites that are apparently required for end-1 activation in the E lineage. Whereas only ~20% of embryos lacking maternal SKN-1 protein make any endoderm, 100% of skn-1 mutant embryos expressing end-1 from the heat shock promoter make large amounts of endoderm; these embryos are indistinguishable from the skn-1(+) embryos expressing the construct. This suggests that a primary function of these maternal factors is to regulate zygotic end-1 expression, which is then sufficient to initiate the entire program for endoderm development (Zhu, 1998).
The Caenorhabditis elegans elt-2 gene encodes a single-finger GATA factor, previously cloned by virtue of its binding to a tandem pair of GATA sites that control the gut-specific ges-1 esterase gene. elt-2 expression is completely gut specific, beginning when the embryonic
gut has only two cells (one cell cycle prior to ges-1 expression) and continuing in every cell of the gut throughout the life of the worm. When elt-2 was expressed ectopically using a transgenic heat-shock construct, the endogenous ges-1 gene was expressed in most if not all cells of the embryo; several
other gut markers (including a transgenic elt-2-promoter::lacZ reporter construct designed to test for elt-2 autoregulation) were also expressed ectopically in the
same experiment. These effects are specific in that two other C. elegans GATA factors (elt-1 and elt-3) do not cause ectopic gut gene expression. An imprecise transposon excision was identified that removes the entire elt-2 coding region. Homozygous elt-2 null mutants die at the L1 larval stage with an apparent malformation or degeneration of gut cells. Although the loss of elt-2 function has major consequences for later gut morphogenesis and function, mutant embryos still express ges-1. It has been suggested that elt-2 is part of a redundant network of genes that controls embryonic gut development; other factors may be able to compensate for elt-2 loss in the earlier stages of gut development but not in later stages (Fukushige, 1998).
The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. These results suggest that a broad tissue
territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network (Maduro, 2001).
In vertebrates, a broad set of cell types is generated from a single tissue territory, mesendoderm. The
mesendoderm becomes subdivided into endoderm and a subset of the mesoderm ('splanchnopleural') that
generates heart and blood. Similarly, in C. elegans, a broad set of cell
types is generated from a single cell, EMS. The EMS lineage becomes subdivided into endoderm (E cell) and a
subset of mesoderm (MS cell) that generates part of the heart-like pharynx and coelomocytes (putative primitive blood cells). Both zebrafish and C. elegans first express a GATA factor (Faust/GATA5 in zebrafish; MED-1,2 in C. elegans) throughout the
mesendoderm prior to gastrulation that is sufficient to direct mesendoderm development in nonmesendodermal
cells. In both C. elegans and vertebrates, a set of conserved regulators acts after each germ layer type has been
segregated from the mesendoderm. These include HNF-3-like and HNF-4-like factors in the endoderm and the cardiac/pharynx-promoting Nkx2.5/CEH-22 factors in the mesoderm. These observations suggest that a conserved gene regulatory network may underly mesendoderm specification in all triploblastic metazoans. The generation of a common progenitor of endoderm and a subset of the mesoderm may reflect a decisive event in metazoan evolution that has been preserved in both a large group of cells in vertebrates and in a single cell in C. elegans (Maduro, 2001).
In ecdysozoan protostomes, including arthropods and nematodes, transcription factors of the GATA family specify the endoderm:
Drosophila dGATAb (ABF/Serpent) and Caenorhabditis elegans END-1 play important roles in generating this primary germ layer.
end-1 is the earliest expressed endoderm-specific gene known in C. elegans and appears to initiate the program of gene expression
required for endoderm differentiation, including a cascade of GATA factors required for development and maintenance of the intestine.
Among vertebrate GATA proteins, the GATA-4/5/6 subfamily regulates aspects of late endoderm development, but a role for GATA
factors in establishing the endoderm is unknown. END-1 is shown to bind to the canonical target DNA sequence WGATAR with specificity similar to that of
vertebrate GATA-1 and GATA-4, and it functions as a transcriptional activator. This activity of END-1 was exploited to demonstrate that establishment of the
vertebrate endoderm, like that of invertebrate species, also appears to involve GATA transcriptional activity. Like the known vertebrate endoderm regulators Mixer and
Sox17, END-1 is a potent activator of endoderm differentiation when expressed in isolated Xenopus ectoderm. Moreover, a dominant inhibitory GATA-binding fusion protein abrogates
endoderm differentiation in intact embryos. By examining these effects in conjunction with those of Mixer- and Sox17beta-activating and dominant inhibitory
constructs, the likely relationships between GATA activity and these regulators in early development of the vertebrate endoderm have been established. These results
suggest that GATA factors may function sequentially to regulate endoderm differentiation in both protostomes and deuterostomes (Shoichet, 2000).
VegT is the best characterized maternally expressed transcription factor known to
regulate Xenopus endoderm development, whereas Mixer and Sox17 alpha and beta are the earliest known zygotic factors. Accordingly,
ectopic expression of VegT in Xenopus ectodermal explants induces expression of both Mixer and Sox17alpha mRNAs. In contrast, END-1 induces
rapid (Nieuwkoop stage 11.5) expression of Sox17alpha but not of Mixer. Thus, both VegT and END-1 activate early expression of Sox17alpha,
implicating GATA function early in endoderm development, and likely upstream of the Sox17 genes. The results further suggest that induction of Sox17alpha by
END-1 is either independent of Mixer, or that the proposed linear pathway from Mixer to Sox17alpha in part involves a GATA-factor intermediary (Shoichet, 2000).
The C. elegans GATA transcription factor genes elt-1 and elt-3 are expressed in the embryonic hypodermis (also
called the epidermis). elt-1 is expressed in precursor cells and is essential for the production of most hypodermal cells. elt-3
is expressed in all of the major hypodermal cells except the lateral seam cells, and expression is initiated immediately after the
terminal division of precursor lineages. Although this expression pattern suggests a role for ELT-3 in hypodermal development, no functional studies have yet been performed. It has been shown that either elt-3 or elt-1 is sufficient, when force expressed in early embryonic blastomeres, to activate a program of hypodermal differentiation even in blastomeres that are not hypodermal
precursors in wild-type embryos. The elt-3 gene has been deleted and ELT-3 has been shown to be not essential for either hypodermal cell differentiation or the viability of the organism. ELT-3 can activate hypodermal gene expression in the absence of ELT-1 and, conversely, ELT-1 can activate
hypodermal gene expression in the absence of ELT-3. Overall, the combined results of the mutant phenotypes, initial expression times, and forced-expression experiments suggest that ELT-3 acts downstream of ELT-1 in a redundant pathway controlling hypodermal cell differentiation (Gilleard, 2001).
In C. elegans, histone acetyltransferase CBP-1 counteracts the repressive activity of the histone deacetylase HDA-1 to allow endoderm differentiation, which is specified by the E cell. In the sister
MS cell, the endoderm fate is prevented by the action of an HMG box-containing protein, POP-1, through an unknown mechanism. CBP-1, HDA-1 and POP-1 converge on end-1, a Serpent-related GATA factor that acts as an initial endoderm-determining gene. In the E lineage, an essential function of CBP-1 appears to be the activation of end-1 transcription. A molecular mechanism has been identified for the endoderm-suppressive effect of POP-1 in the MS lineage by demonstrating that POP-1 functions as a transcriptional repressor that inhibits inappropriate end-1 transcription. Evidence is provided that POP-1 represses transcription via the recruitment of HDA-1 and UNC-37, the C. elegans homolog of the co-repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate-determining gene during development. Furthermore, they identify a strategy by which concerted actions of histone deacetylases and other co-repressors ensure maximal repression of inappropriate cell type-specific gene transcription (Calvo, 2001).
Development of the vulva in C. elegans is mediated by the
combinatorial action of several convergent regulatory inputs, three of which
(the Ras, Wnt and Rb-related pathways) act by regulating expression of the
lin-39 Hox gene. LIN-39 specifies cell fates and regulates cell
fusion in the mid-body region, leading to formation of the vulva. In the
lateral seam epidermis, differentiation and cell fusion have been shown to be
regulated by two GATA-type transcription factors, ELT-5 and -6.
ELT-5 is encoded by the egl-18 gene, which
promotes formation of a functional vulva. Furthermore, EGL-18
(ELT-5), and its paralogue ELT-6, are redundantly required to regulate cell
fates and fusion in the vulval primordium and are essential to form a vulva.
Elimination of egl-18 and elt-6 activity results in arrest
by the first larval stage; however, in animals rescued for this larval
lethality by expression of ELT-6 in non-vulval cells, the post-embryonic cells
(P3.p-P8.p) that normally become vulval precursor cells often fuse with the
surrounding epidermal syncytium or undergo fewer than normal cell divisions,
reminiscent of lin-39 mutants. Moreover, egl-18/elt-6
reporter gene expression in the developing vulva is attenuated in
lin-39(rf) mutants, and overexpression of egl-18 can
partially rescue the vulval defects caused by reduced lin-39
activity. LIN-39/CEH-20 heterodimers bind two consensus HOX/PBC sites in a
vulval enhancer region of egl-18/elt-6, one of which is essential for
vulval expression of egl-18/elt-6 reporter constructs. These findings
demonstrate that the EGL-18 and ELT-6 GATA factors are essential, genetically
redundant regulators of cell fates and fusion in the developing vulva and are
apparent direct transcriptional targets of the LIN-39 Hox protein (Kohl, 2002).
Hox proteins appear to require co-factors to achieve DNA-binding
specificity. The most extensively studied of the Hox co-factor genes are
the Drosophila extradenticle (exd) and mammalian pre-B
cell homeobox 1 genes, collectively referred to as PBC genes. Hox and PBC
proteins form heterodimers that bind DNA in vitro. C. elegans
contains one known Exd homolog, CEH-20, which appears to act as a Hox
co-factor. Consistent with the possibility that egl-18 and
elt-6 are direct targets of LIN-39 Hox, several consensus
Hox/PBC-binding sites (TGATNNAT) were found in the egl-18 and elt-6 genomic region. Two of these [site 1 (TGATATAT) and site 2 (TGATTGAT)] are present in intron 2 of egl-18, which is included in the ~800 bp promoter element that directs GFP expression in the VPC lineages and VC neurons. Several lines of evidence indicate that site 1, but not site 2, is important for vulval-specific expression of egl-18/elt-6: (1) alteration of 6 bp in site 1 eliminates expression in the VPC lineages and VC neurons, whereas a similar mutation that alters 4 bp of site 2 has no obvious effect on reporter expression; (2) a reporter in which 544 base pairs surrounding only site 1 is present showed expression in the vulva and VC neurons, albeit at an attenuated level compared with the reporter containing both sites; (3) mutation of site 1 from this construct eliminated vulval and VC expression, and (4) comparison of the egl-18 sequence of C. elegans and C. briggsae revealed a highly conserved 27 bp element surrounding Site 1 but no conservation of site 2. Thus, the site 1 Hox/PBC site is apparently necessary and sufficient for vulva-specific expression of
egl-18/elt-6::GFP (Kohl, 2002).
Electrophoretic mobility shift assays were performed to test the hypothesis
that egl-18 and elt-6 are direct targets of LIN-39/CEH-20
heterodimers in the vulva. Indeed, LIN-39 and CEH-20
heterodimers bind in vitro to 30 bp oligonucleotides centered on either the
Hox/PBC site 1 or site 2. Whereas binding of LIN-39/CEH-20 to site 1 oligos could be competed away with excess unlabeled site 1 or 2 oligos, unlabeled site 1 oligos could not compete with site 2 oligos, implying that site 2
has a higher in vitro affinity for LIN-39/CEH-20 than does site 1. These results indicate that LIN-39/CEH-20 heterodimers can bind cooperatively to site 1, which is essential for expression of the egl-18/elt-6 reporter in the vulva. Based on these results and the phenotypes of egl-18/elt-6
mutants, it seems likely that LIN-39 regulates vulval development by directly
activating EGL-18 and ELT-6, which in turn repress epidermal fusion and
activate vulval differentiation (Kohl, 2002).
The elt-4 gene from the nematode C. elegans is predicted to encode a very small (72 residues, 8.1 kD) GATA-type zinc finger transcription factor. The elt-4 gene is located ~5 kb upstream of the C. elegans elt-2 gene, which also encodes a GATA-type transcription factor; the zinc finger DNA-binding domains are highly conserved (24/25 residues) between the two proteins. The elt-2 gene is expressed only in the intestine and is essential for normal intestinal development. This article explores whether elt-4 also has a role in intestinal development. Reporter fusions to the elt-4 promoter or reporter insertions into the elt-4 coding regions show that elt-4 is indeed expressed in the intestine, beginning at the 1.5-fold stage of embryogenesis and continuing into adulthood. elt-4 reporter fusions are also expressed in nine cells of the posterior pharynx. Ectopic expression of elt-4 cDNA within the embryo does not cause detectable ectopic expression of biochemical markers of gut differentiation; furthermore, ectopic elt-4 expression neither inhibits nor enhances the ectopic marker expression caused by ectopic elt-2 expression. A deletion allele of elt-4 was isolated but no obvious phenotype could be detected, either in the gut or elsewhere; brood sizes, hatching efficiencies, and growth rates were indistinguishable from wild type. No evidence was found that elt-4 provided backup functions for elt-2. Microarray analysis was used to search for genes that might be differentially expressed between L1 larvae of the elt-4 deletion strain and wild-type worms. Paired hybridizations were repeated seven times, leading to the conclusion that no candidate target transcript could be identified as significantly up- or down-regulated by loss of elt-4 function. In vitro binding experiments could not detect specific binding of ELT-4 protein to candidate binding sites (double-stranded oligonucleotides containing single or multiple WGATAR sequences); ELT-4 protein neither enhanced nor inhibited the strong sequence-specific binding of the ELT-2 protein. Whereas ELT-2 protein is a strong transcriptional activator in yeast, ELT-4 protein has no such activity under similar conditions, nor does it influence the transcriptional activity of coexpressed ELT-2 protein. Although an elt-2 homolog was easily identified in the genomic sequence of the related nematode C. briggsae, no elt-4 homolog could be identified. Analysis of the changes in silent third codon positions within the DNA-binding domains indicates that elt-4 arose as a duplication of elt-2, some 25-55 MYA. Thus, elt-4 has survived far longer than the average duplicated gene in C. elegans, even though no obvious biological function could be detected. elt-4 provides an interesting example of a tandemly duplicated gene that may originally have been the same size as elt-2 but has gradually been whittled down to its present size of little more than a zinc finger. Although elt-4 must confer (or must have conferred) some selective advantage to C. elegans, it is suggested that its ultimate evolutionary fate will be disappearance from the C. elegans genome (Fukushige, 2003).
Mesoderm and endoderm in C. elegans arise from sister cells called MS and E,
respectively. The identities of both of these mesendodermal progenitors are
controlled by MED-1 and -2, members of the GATA factor family. In the E lineage,
these factors activate a sequential cascade of GATA factors, beginning with
their immediate targets, the endoderm-specifying end genes. MED-1
binds invariant noncanonical sites in the end genes, revealing that the MEDs are
atypical members of the GATA factor family that do not recognize GATA sequences.
By searching the genome for clusters of these MED sites, 19
candidate MED targets were identified. Based on their expression patterns, these define three
distinct classes of MED-regulated genes: MS-specific, E-specific, and E plus
MS-specific. Some MED targets encode transcription factors related to those that
regulate mesendoderm development in other phyla, supporting the existence of an
ancient metazoan mesendoderm gene regulatory network (Broitman-Maduro, 2005).
To define the C. elegans aging process at the molecular level, DNA microarray experiments were used to identify a set of 1294 age-regulated genes and it was found that the GATA transcription factors ELT-3, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-3, which shows a corresponding decrease in expression in old worms. elt-3 regulates a large number of downstream genes that change expression in old age, including ugt-9, col-144, and sod-3. elt-5(RNAi) and elt-6(RNAi) worms have extended longevity, indicating that elt-3, elt-5, and elt-6 play an important functional role in the aging process. These results identify a transcriptional circuit that guides the rapid aging process in C. elegans and indicate that this circuit is driven by drift of developmental pathways rather than accumulation of damage (Budovskaya, 2008).
A widely held view is that aging is caused by accumulation of damage, and, thus, one might expect that age-related changes in the elt-3 transcriptional network would be caused by a lifelong accumulation of damage or stress. In mammals, there is abundant evidence that aging is the result of damage accumulation, such as oxidative damage, somatic DNA mutation, telomere shortening, protein glycation, and inflammation. However, worms age very rapidly compared to mammals, and it is unclear whether the rate of damage accumulation is high enough to account for the short worm life span. No evidence was found that age regulation of the elt-3 transcriptional network is caused by accumulation of damage, stress, or inflammation (Budovskaya, 2008).
Besides damage accumulation, another possibility is that aging might result from developmental pathways that go awry late in life (antagonistic pleiotropy). In worms, it was found that decreased expression of elt-3 GATA in old age is caused by increased expression of elt-5 GATA or elt-6 GATA, which act as repressors. The activities of elt-5 or elt-6 are not known to be affected by cellular damage or environmental stressors, and, thus, drift in the GATA transcriptional hierarchy might be due to intrinsic processes. However, it cannot be completely ruled out that age-related changes in the elt-3/elt-5/elt-6 GATA transcriptional circuit are caused by extrinsic factors, and further work on the nature of age-related changes will help resolve this issue (Budovskaya, 2008).
How could the elt-3/elt-5/elt-6 transcriptional network for aging evolve? It seems unlikely that any changes in old age could provide a selective advantage and be under natural selection. In the wild, worms usually die of predation rather than old age, and traits that are only evident in old worms would have little effect on fitness. Rather than evolving under the force of natural selection, another possibility is that age-related changes in the elt-3/elt-5/elt-6 transcriptional network have a neutral effect on fitness in the wild and have become fixed in the C. elegans genome (the mutation accumulation theory). Regulation of elt-3 by elt-5 and elt-6 would be under evolutionary selection because of its important early role during development. In old worms, there is little or no advantage to maintaining proper elt-3 expression, and decreased expression of elt-3 might occur as a secondary consequence. This could shorten the life of old worms but would have a neutral effect on population fitness, as old worms are extremely rare in the wild. Thus, the elt-3/elt-5/elt-6 hierarchy is a developmental program that may change during aging simply because proper homeostatic maintenance in late life is not under the force of natural selection (Budovskaya, 2008).
Gene expression profiles for aging have been defined in many other animals, including flies, mice, and humans. It will be interesting to determine whether transcriptional changes during aging in other animals are also caused by imbalances in developmental regulatory hierarchies (Budovskaya, 2008).
Terminally differentiated post-mitotic cells are generally considered irreversibly developmentally locked, i.e. incapable of being reprogrammed in vivo into entirely different cell types. Brief expression of a single transcription factor, the ELT-7 GATA factor, can convert the identity of fully differentiated, highly specialized non-endodermal cells of the pharynx into fully differentiated intestinal cells in intact larvae and adult Caenorhabditis elegans. Stable expression of intestine-specific molecular markers parallels loss of markers for the original differentiated pharynx state; hence, there is no apparent requirement for a dedifferentiated intermediate during the transdifferentiation process. Based on high-resolution morphological characteristics, the transdifferentiated cells become remodeled to resemble typical intestinal cells at the level of both the cell surface and internal organelles. Thus, post-mitotic cells, though terminally differentiated, remain plastic to transdifferentiation across germ layer lineage boundaries and can be remodeled to adopt the characteristics of a new cell identity without removal of inhibitory factors. These findings establish a simple model to investigate how cell context influences forced transdifferentiation of mature cells (Riddle, 2013).
Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 (homolog of Drosophila Sex-combs reduced) is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, transcription factors necessary for lin-39 expression in the VPCs, factors were sought that initiate lin-39 expression in the embryo. The yeast one-hybrid (Y1H) method was used to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and several factors were characterized by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site that was identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs. These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust (Liu, 2014).
Invertebrate defence against pathogens exclusively relies on components of the innate immune system. Comprehensive information has been collected over the last years on the molecular components of invertebrate immunity and the involved signalling processes, especially for the main invertebrate model species, the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans. Yet, the exact regulation of general and specific defences is still not well understood. In the current study, advantage was taken of a recently established database, WormExp, which combines all available gene expression studies for C. elegans, in order to explore commonalities and differences in the regulation of nematode immune defence against a large variety of pathogens versus food microbes. Significant overlaps were identified in the transcriptional response towards microbes, especially pathogenic bacteria. It was also found that the GATA motif is overrepresented in many microbe-induced gene sets and in targets of other previously identified regulators of worm immunity. Moreover, the activated targets of one of the known C. elegans GATA transcription factors, ELT-2 (see Drosophila Serpent), are significantly enriched in the gene sets, which are differentially regulated by gut-infecting pathogens. These findings strongly suggest that GATA transcription factors and particularly ELT-2 play a central role in regulating the C. elegans immune response against gut pathogens. More specific responses to distinct pathogens may be mediated by additional transcription factors, either acting alone or jointly with GATA transcription factors. Taken together, this analysis of the worm's transcriptional response to microbes provides a new perspective on the C. elegans immune system, which is proposed to be coordinated by GATA transcription factor ELT-2 in the gut (Yang, 2016).
In chordates, formation of neural tissue from ectodermal cells requires an induction. The molecular nature of the inducer remains controversial in vertebrates. Using the early neural marker Otx as an entry point, the neural induction pathway in the simple embryos of Ciona intestinalis was dissected. The regulatory element driving Otx expression in the prospective neural tissue was isolated; this element directly responds to FGF signaling and FGF9/16/20 acts as an endogenous neural inducer. Binding site analysis and gene loss of function established that FGF9/16/20 induces neural tissue in the ectoderm via a synergy between two maternal response factors. Ets1/2 mediates general FGF responsiveness, while the restricted activity of GATAa targets the neural program to the ectoderm. Thus, this study identifies an endogenous FGF neural inducer and its early downstream gene cascade. It also reveals a role for GATA factors in FGF signaling (Bertrand, 2003).
Otx expression starts in the animal a6.5 pair of blastomeres as they become restricted to anterior neural fate, at the onset of the neural induction process. At this stage, Otx is also activated in the animal b6.5 pair of blastomeres (precursors of the posterior dorsal neural tube and of the dorsal midline which constitutes a neurogenic region and in some vegetal B-line blastomeres (precursors of the posterior mesendoderm). Interestingly, Otx activation in b6.5, as in a6.5, requires an induction from vegetal blastomeres (Bertrand, 2003 and references therein).
The region in Otx located between -1541 and -1417 is required for expression in the a6.5 lineage, and is referred to as the a-element. Consistent with the simultaneous induction of Otx in a6.5 and b6.5 by vegetal cells, deletion of the a-element also reduces the activity in the b6.5 lineage. Finally, regions located between positions -1417 to -1133, and -706 to -271 are required for expression in A-line, and B/b-lines respectively (Bertrand, 2003).
Otx activation in the a6.5 neural precursors requires an interaction with the anterior vegetal blastomeres (A-line). Thus, the inducing FGF should be expressed in A-line blastomeres, before the onset of Otx expression at the 32-cell stage. The Ciona intestinalis genome contains 6 members of the FGF family. By in situ hybridization, only detect one FGF, FGF9/16/20, could be detected that was expressed at the right time and place to be the inducer. Its expression starts at the 16 cell-stage in the A-line and some B-line cells. Expression is stronger in the A-line than in the B-line, and this difference is further enhanced at the early 32-cell stage. This expression pattern is similar to that of the Ciona savignyi ortholog and is consistent with a role for FGF9/16/20 as endogenous neural inducer (Bertrand, 2003).
By both gene loss of function and binding sites analysis it has been determined that cooperation between the maternal transcription factors, Ets1/2 and GATAa, mediates the initial transcriptional response to FGF. Ets transcription factors have already been shown to act in the FGF pathway in vertebrates, and the members of the Ets1/2 subfamily can be directly phosphorylated and activated by Erk. A role for GATAa in this process was more unexpected, since GATA factors have so far not been implicated in the FGF pathway. However, the fact that multimerized GATA binding sites mediate FGF responsiveness indicate that, in this system, GATA does not act solely to modify or enhance Ets activity but functions as an FGF-activated transcription factor. Consistent with the proposal of a direct involvement of GATA factors in the FGF pathway in vivo, it has recently been shown, in vitro, that vertebrate GATA4 can be directly phosphorylated and activated by Erk (Bertrand, 2003).
Could members of the Ets1/2 and GATA families also play a role in neural induction in vertebrates? Ets2 messenger is present maternally in Xenopus eggs and has recently been shown to be required for the induction of Brachyury by FGF in mesodermal cells. It will be interesting to test whether it also acts in the neural induction pathway. Vertebrate GATA factors are thought to antagonize rather than promote neural tissue formation; GATA1/2/3 family members are expressed during gastrulation in the nonneural ectoderm in zebrafish, Xenopus, and chick and GATA1 has an antineuralizing activity when overexpressed in Xenopus. However, GATA2 has no antineuralizing activity, showing that this is not a general property of GATA factors. GATA2 and GATA5 are present in Xenopus eggs but the early role of these maternal GATA factors has not been studied, leaving open the possibility of an involvement in neural induction. Finally, it is proposed that, in ascidians, the use of different response factors accounts for the activation of different target genes in neuroectoderm and mesoderm. It will be interesting to test whether the same logic is used in vertebrates or whether the increase in gene number has led to the recruitment of different FGF inducers or receptors in these two lineages (Bertrand, 2003 and references therein).
Inderstanding of the maternal factors that initiate early chordate development, and of their direct zygotic targets, is still fragmentary. A molecular cascade is emerging for the ascidian mesendoderm, but less is known about the ectoderm, giving rise to epidermis and nervous tissue. Cis-regulatory analysis surprisingly places the maternal transcription factor Ci-GATAa (GATA4/5/6) at the top of the ectodermal regulatory network in ascidians. Initially distributed throughout the embryo, Ci-GATAa activity is progressively repressed in vegetal territories by accumulating maternal β-catenin. Once restricted to the animal hemisphere, Ci-GATAa directly activates two types of zygotic ectodermal genes. First, Ciona friend of GATA gene (Ci-fog) is activated from the 8-cell stage throughout the ectoderm, then Ci-otx is turned on from the 32-cell stage in neural precursors only. Whereas the enhancers of both genes contain critical and interchangeable GATA sites, their distinct patterns of activation stem from the additional presence of two Ets sites in the Ci-otx enhancer. Initially characterized as activating elements in the neural lineages, these Ets sites additionally act as repressors in non-neural lineages, and restrict GATA-mediated activation of Ci-otx. This study has identified a precise combinatorial code of maternal factors responsible for zygotic onset of a chordate ectodermal genetic program (Rothbacher, 2007).
GATA-1: General considerations Serpent is a member of the GATA family of transcription factors. The four members of the GATA-family of vertebrates all contain two zinc fingers, both of which are necessary for DNA binding (Abel, 1993). The second GATA homolog in Drosophila is pannier, also known as GATA-2.
In vertebrates, transcriptional regulators of the GATA family appear to have a conserved function in differentiation and organ development. GATA-1, -2 and -3 are required for different aspects of hematopoiesis, while GATA-4, -5 and -6 are expressed in various organs of endodermal origin, such as intestine and liver, and are implicated in endodermal differentiation. GATA-1 is required for primitive and definitive erythropoiesis, GATA-2 for early hematopoiesis, and GATA-3 is implicated in the differentiation of T-lymphocytes. Pannier and Serpent are recently diverged on an evolutionary scale, each similarly related in sequence to all vertebrate GATA proteins (Rehorn, 1996 and references).
DNA binding of GATA transcription factors is mediated through two domains, each containing a zinc finger. Although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. These results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo (Pedone, 1997).
The ventralizing factor bone morphogenetic protein 4 (BMP-4, Drosophila homolog: Decapentaplegic) can inhibit Xenopus neurogenesis. The erythroid transcription factor GATA-1 functions downstream of the BMP-4 signaling pathway and mediates BMP-4-induced erythropoiesis. Similar to BMP-4, GATA-1b inhibits neuralization of Xenopus animal cap (AC) cells. The neural inhibition is not seen with GATA-1a, although both GATA-1a and GATA-1b RNAs are translated at the same efficiency and induce globin expression equally in AC cells. GATA-1b RNA injection into AC cells neither induces expression of Xbra (a general mesoderm marker) nor affects expression of XK81 (epidermal keratin) or BMP-4 and Xvent-1 (two ventral markers). These data suggest that GATA-1b retains the epidermal fate of the AC. Intact GATA-1b protein is required for both inhibition of neurogenesis and induction of globin expression. These findings indicate that GATA-1b can function in ectoderm to specifically regulate neural inducing mechanisms, apparently related to the expression of chordin, a neuralizing gene. Tadpole stage embryos injected with GATA-1b are devoid of all dorsoanterior structures including neural tissue. This report provides evidence that the two transcription factors, derived from a recent genome duplication, share a common biological activity (stimulation of erythropoiesis) while also exhibiting a distinct function; the inhibition of neurogenesis (Xu, 1997).
The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, an erythroid cell line was generated from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. Rescue of terminal erythroid maturation was used in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences have been established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. It is proposed that a nuclear cofactor interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, this experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function (Weiss, 1997).
In nonhematopoietic cells, CREB-binding protein (CBP) markedly stimulates GATA-1's transcriptional activity in transient transfection experiments. GATA-1 and CBP also coimmunoprecipitate from nuclear extracts of erythroid cells. Interaction mapping pinpoints contact sites to the zinc finger region of GATA-1 and to the E1A-binding region of CBP. Expression of a conditional form of adenovirus E1A in murine
erythroleukemia cells blocks differentiation and expression of endogenous GATA-1 target genes,
whereas mutant forms of E1A, unable to bind CBP/p300, have no effect. These findings add GATA-1,
and very likely other members of the GATA family, to the growing list of molecules implicated in the
complex regulatory network surrounding CBP/p300 (Blobel, 1998).
The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. The transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro does not alter the ability of GATA-1 to bind DNA, and mutations in either motif do not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. Mutations in either acetylation motif partially impairs the ability of GATA-1 to induce differentiation while mutations in both motifs abrogate it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells (Hung, 1999).
Mammalian SWI/SNF chromatin remodeling complexes are involved in critical aspects of cellular growth and genomic stability. Each complex contains one of two highly homologous ATPases, BRG1 and BRM, yet little is known about their specialized functions. BRG1 and BRM are shown to associate with different promoters during cellular proliferation and differentiation, and in response to specific signaling pathways by preferential interaction with certain classes of transcription factors. BRG1 binds to zinc finger proteins through a unique N-terminal domain that is not present in BRM. BRM interacts with two ankyrin repeat proteins that are critical components of Notch signal transduction. Thus, BRG1 and BRM complexes may direct distinct cellular processes by recruitment to specific promoters through protein-protein interactions that are unique to each ATPase (Kadam, 2003).
SWI/SNF interacts with zinc finger proteins (ZFP)s through the ZF DNA-binding domain (DBD) and the BRG1 ATPase. The basis for the observed specificity between ZFPs and BRG1 complexes is that interaction occurs within a domain of BRG1 that is nonhomologous with BRM. The role of individual ZFs within two structural motifs, C2H2 and C4, was investigated in mediating BRG1 SWI/SNF function. Using the erythroid factors EKLF and GATA-1 as representative proteins that contain C2H2 and C4 domains, respectively, these studies demonstrate that BRG1 binds to individual ZFs that are the most critical for DNA binding. This may seem paradoxical; however, ZF DBDs have been shown to associate with both RNA and protein. The EKLF and GATA-1 DBDs interact with a variety of cofactors, often through specific ZFs. The significance of such critical protein-protein interactions, including that of SWI/SNF, occurring through domains that must also bind DNA has yet to be elucidated. The functional relationship between BRG1-containing SWI/SNF and the ZF DBDs of EKLF or GATA-1 may pertain to other members of these transcription factor families which contain conserved DBDs but also contain highly divergent activation domains which contribute to their specialized functions in gene regulation (Kadam, 2003).
The BRM ATPase is expressed at high levels in differentiating cells, yet the functional role of this protein and the identity of the genes it regulates are poorly understood. In this regard, the observation that two components of the Notch signaling pathway, CBF-1 and ICD22 (the intracellular domain of Notch), strongly associate with BRM but not BRG1 is especially intriguing. This pathway controls cell fate commitment in a broad range of developmental processes. CBF-1 recruits BRM to two natural target genes, Hes1 and Hes5, in myoblasts before Notch induction. This indicates that these promoters are already in a remodeled configuration and accessible to bind the activator, Notch2, upon signaling (Kadam, 2003).
Gfi-1B (growth factor independence-1B) gene is an erythroid-specific transcription factor, whose expression plays an essential role in erythropoiesis. The human Gfi-1B promoter region has been defined; GATA-1 mediates erythroid-specific Gfi-1B transcription. By further investigating the regulation of the Gfi-1B promoter, this study reports that (1) Gfi-1B transcription is negatively regulated by its own gene product, (2) GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the Gfi-1B promoter and (3) Gfi-1B suppresses GATA-1-mediated stimulation of Gfi-1B promoter through their protein interaction. These results not only demonstrate that interaction of GATA-1 and Gfi-1B participates in a feedback regulatory pathway in controlling the expression of the Gfi-1B gene, but also provide the first evidence that Gfi-1B can exert its repression function by acting on GATA-1-mediated transcription without direct binding to the Gfi-1 site of the target genes. Based on these data, it is proposed that this negative auto-regulatory feedback loop is important in restricting the expression level of Gfi-1B, thus optimizing its function in erythroid cells (Huang, 2005).
GATA-1 and erythroid maturation DNA constructs containing the putative zebrafish promoter sequences of
GATA-1, an erythroid-specific transcription factor, and the green fluorescent protein reporter gene, were microinjected into single-cell zebrafish embryos.
Erythroid-specific activity of the GATA-1 promoter is observed in living embryos
during early development. Fluorescent circulating blood cells are detected in
microinjected embryos 24 hours after fertilization and are still present in 2-month-old fish. Germline transgenic fish obtained from the injected founders continue to express green fluorescent protein in erythroid cells in the F1 and F2 generations. The green fluorescent protein expression patterns in transgenic fish are consistent with the pattern of GATA-1 mRNA expression detected by RNA in situ hybridization. These transgenic fish have allowed for the isolation, by fluorescence-activated cell sorting, of the earliest erythroid progenitor cells from developing embryos, making in vitro studies possible. The earliest progenitor cells are larger, more brightly stained and less abundant than other GFP expressing circulating blood cells. Preliminary experiments show that they consist of cells that have long-term proliferative capacity (Long, 1997).
Expression of gata1 is regulated through multiple cis-acting GATA motifs. Zebrafish has been used to elucidate regulatory mechanisms of the gata1 gene. To this end, zebrafish gata1 genomic DNA was isolated and analyzed. This resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA was microinjected into embryos. Surprisingly, ectopic expression of the reporter gene is induced at the site of GATA1 overexpression and is dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct reveal that both the N- and C-terminal zinc-finger domains of GATA1, that is intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation (Kobayashi, 2001).
The zinc-finger transcription factor GATA-1 binds to GATA consensus elements in regulatory regions of the alpha- and beta-globin gene clusters and other erythroid cell-specific genes. Analysis of the effects of mutations in GATA-binding sites in cell culture and in binding assays in vitro, as well as transactivation studies with GATA-1 expression vectors in heterologous cells, have provided indirect evidence that this factor is involved in the activation of globin and other genes during erythroid cell maturation. GATA-1 is also expressed in megakaryocytes and mast cells, but not in other blood cell lineages or in non-hemopoietic cells. To investigate the role of this factor in hematopoiesis in vivo, the X-linked GATA-1 gene was disrupted by homologous
recombination in a male (XY) murine embryonic stem cell line and the GATA-1-deficient cells were tested for their ability to contribute to different tissues in chimeric mice. The mutant embryonic stem cells contribute to all non-hemopoietic tissues tested and to a white blood cell fraction, but fail to give rise to mature red blood cells. This demonstrates that GATA-1 is required for the normal differentiation of erythroid cells, and that other GATA-binding proteins cannot compensate for its absence (Pevney, 1991).
GATA-1 is a zinc-finger transcription factor believed to play an important role in gene regulation during the development of erythroid cells, megakaryocytes and mast cells. Other members of the GATA family, which can bind to the same DNA sequence motif, are co-expressed in several of these hemopoietic lineages, raising the possibility of overlap in function. In vitro colony assays were used to identify the stage at which mutant erythroid cells are affected by lack of GATA-1, and to examine the requirement for GATA-1 in other lineages. The development of erythroid progenitors in embryonic yolk sacs is unaffected by GATA-1 mutation, but cells failed to mature beyond the proerythroblast stage, an early point in terminal differentiation. GATA-1- colonies contain phenotypically normal macrophages, neutrophils and megakaryocytes, indicating that GATA-1 is not required for the in vitro differentiation of cells in these lineages. GATA-1- megakaryocytes are abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. The lack of a block in terminal megakaryocyte differentiation was shown by the in vivo production of platelets expressing the ES cell-derived GPI-1C isozyme. The role of GATA-1 in mast cell differentiation was examined by the isolation of clonal mast cell cultures from chimeric fetal livers. Mutant and wild-type mast cells display similar growth and histochemical staining properties after culture under conditions that promote the differentiation of cells resembling mucosal or serosal mast cells. Thus, the mast and megakaryocyte lineages, in which GATA-1 and GATA-2 are co-expressed, can complete their maturation in the absence of GATA-1, while erythroid cells, in which GATA-1 is the predominant GATA factor, are blocked at a relatively early stage of maturation (Pevny, 1995).
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, a novel, multitype zinc finger protein, Friend of GATA-1 (FOG) has been identified. FOG (Drosophila homolog: U-shaped) binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG has nine zinc finger motifs distributed throughout the protein. Four of the fingers are of the C2H2 type, while the GATA-1 interaction region encompasses a single C2HC finger. Two adjacent C2H2 fingers in FOG resemble double zinc fingers found in two developmental proteins, the Drosophila homeoprotein Spalt and the C. elegans protein SEM-4. 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 (Tsang, 1997).
Transcription factors of the GATA-family are essential for proper development of diverse tissues and cell types. GATA-1 is required for differentiation of two hematopoietic lineages (red blood cells and megakaryocytes), whereas GATA-3 is essential for T-cell development. Functional studies suggest that many properties of the GATA-family of proteins are shared and largely interchangeable. To test whether the function of GATA-1 in erythroid differentiation can be replaced by another GATA-factor, a knock-in mutation of the GATA-1 locus was generated in which GATA-3 cDNA was introduced by gene targeting. Mutant embryos, though embryonic lethal, exhibit partial rescue, characterized by increased survival of erythroid precursor cells and improved hemoglobin production. The basis for the incomplete extent of rescue is likely to be complex, but may be accounted for, in part, by insufficient accumulation of GATA-3 protein (compared with the normal level of GATA-1). These findings suggest that GATA-3 protein is functional when expressed in an erythroid environment and is competent to act on at least a subset of erythroid-expressed target genes in vivo (Tsai, 1998).
Protein-protein interactions play significant roles in the control of gene expression. These interactions often occur between small, discrete domains within different transcription factors. In particular, zinc fingers, usually regarded as DNA-binding domains, are now also known to be involved in mediating contacts between proteins. The interaction between the erythroid transcription factor GATA-1 and its partner, the 9 zinc finger protein, FOG (Friend Of GATA), has been investigated. This interaction represents a genuine finger-finger contact, which is dependent on zinc-coordinating residues within each protein. The contact domains have been mapped to the core of the N-terminal zinc finger of GATA-1 and the 6th zinc finger of FOG. Using a scanning substitution strategy, key residues within the GATA-1 N-finger that are required for FOG binding have been identified. These residues are conserved in the N-fingers of all GATA proteins known to bind FOG, but are not found in the respective C-fingers. This observation may, therefore, account for the particular specificity of FOG for N-fingers. Interestingly, the key N-finger residues are seen to form a contiguous surface, when mapped onto the structure of the N-finger of GATA-1 (Fox, 1998).
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 (which 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 the sixth finger of FOG-1 is known to interact specifically with the N-finger but not the C-finger of
GATA-1. Fingers 1, 5 and 9 of FOG-1, all atypical Cys-Cys:His-Cys fingers, also interact with the N-finger of GATA-1;
FOG-2 and U-shaped also contain multiple GATA-interacting fingers and both
FOG-2 and U-shaped contain several Cys-Cys:His-Cys zinc fingers. The key contact
residues are defined and these residues are shown to be highly conserved in GATA-interacting fingers. The
effects of selectively mutating the four interacting fingers of FOG-1 were examined and each is shown to contribute to
FOG-1's ability to modulate GATA-1 activity. FOG-1 can repress GATA-1-mediated
activation: evidence is presented that this ability involves the recently described CtBP co-repressor proteins
that recognize all known FOG proteins (Fox, 1999).
The mechanism by which FOG-1 acts to repress GATA-mediated transcription was investigated. FOG-1 contains a
motif that is bound by the CtBP family of co-repressors. This site PIDLSKR occurs immediately N-terminal to finger 7. Yeast two-hybrid and GST pull-down assays were used to test whether a small region of FOG-1 (residues 724-834, spanning the CtBP-binding motif) could interact with
one family member, mCtBP2. GST-FOG-1(724-834) can retain in vitro-translated mCtBP2 efficiently, whereas a mutant FOG-1
containing a mutation in the core region (PIDLSKR to AIAASKR) is unable to retain mCtBP2. Similarly, in the yeast two-hybrid system,
FOG-1(724-834) is able to interact with mCtBP2, whereas the mutant cannot.
To test if this region of FOG-1 can act as a repression domain in vivo, fusions of FOG-1(724-834) (both wild-type and mutant) with the Gal4
DNA-binding domain were prepared and these were co-transfected with a construct harbouring a Gal4-dependent promoter upstream of the human growth hormone reporter
gene. Gal4DBD-FOG-1(724-834) represses the basal reporter activity 20-fold. However, the mutant is unable to significantly repress transcription. This result indicates that FOG-1 contains a repression domain that can mediate repression by associating with CtBP family proteins (Fox, 1999).
The RBTN2 LIM-domain protein (See Drosophila Muscle LIM protein at 60A), originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. Direct interaction of the RBTN2 protein during erythropoiesis was observed in vivo and in vitro with the GATA1 or GATA2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation (Osada, 1995).
The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukemias, is normally
expressed in hematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the
interaction is unexplained. In erythroid cells Lmo2 forms a novel DNA-binding
complex with GATA-1, TAL1, E2A, and the recently identified LIM-binding protein, Ldb1/NLI (Drosophila homolog: Chip). This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box (CAGGTG), followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding complex, which may play a role in hematopoiesis (Wadman, 1997).
Modification of histones (DNA-binding proteins found in chromatin) by addition of acetyl groups occurs
to a greater degree when the histones are associated with transcriptionally active DNA. A
breakthrough in understanding how this acetylation is mediated was the discovery that various
transcriptional co-activator proteins have intrinsic histone acetyltransferase activity (for example,
Gcn5p, PCAF, TAF(II)250 and p300/CBP. These acetyltransferases also modify certain transcription
factors (TFIIEbeta, TFIIF, EKLF and p53). GATA-1 is an important transcription factor in the
hematopoietic lineage and is essential for terminal differentiation of erythrocytes and megakaryocytes.
It is associated in vivo with the acetyltransferase p300/CBP. GATA-1 is
acetylated in vitro by p300. This significantly increases the amount of GATA-1 bound to DNA and
alters the mobility of GATA-1-DNA complexes. This is suggestive of a conformational change in GATA-1.
GATA-1 is also acetylated in vivo and acetylation directly stimulates GATA-1-dependent transcription.
Mutagenesis of important acetylated residues shows that there is a relationship between the acetylation
and in vivo function of GATA-1. It is proposed that acetylation of transcription factors can alter
interactions between these factors and DNA and among different transcription factors, and is an
integral part of the transcription and differentiation processes (Boyes, 1998).
Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL (related protein, Drosophila Helix loop helix protein 3B) and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. The Xenopus homolog of LMO-2 has been cloned and it has been shown to be
expressed in a pattern similar to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act
synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads
to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis (Mead, 2001).
GATA-1 is a transcription factor essential for erythroid/megakaryocytic cell differentiation. To investigate the contribution of individual domains of GATA-1 to its activity, transgenic mice expressing either an N-terminus, or an N- or C-terminal zinc finger deletion of GATA-1 (DeltaNT, DeltaNF or DeltaCF, respectively) were generated and crossed to GATA-1 germline mutant (GATA-1.05) mice. Since the GATA-1 gene is located on the X-chromosome, male GATA-1 mutants die by embryonic day 12.5. Both DeltaNF and DeltaCF transgenes fail to rescue the GATA-1.05/Y pups. However, transgenic mice expressing DeltaNT, but not the DeltaNF protein, were able to rescue definitive hematopoiesis. In embryos, while neither the DeltaCF protein nor a mutant missing both N-terminal domains (DeltaNTNF) was able to support primitive erythropoiesis, the two independent DeltaNT and DeltaNF mutants could support primitive erythropoiesis. Thus, lineage-specific transgenic rescue of the GATA-1 mutant mouse reveals novel properties that are conferred by specific domains of GATA-1 during primitive and definitive erythropoiesis, and demonstrate that the NT and NF moieties lend complementary, but distinguishable properties to the function of GATA-1 (Shimizu, 2001).
Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, the novel approach has been taken of investigating whether the ability of embryonic stem
(ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. Conditions were established whereby EBs can form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed
hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally
differentiated ß-like globin markers. Using this system, it has been determined that EB differentiation in BMP4 is necessary and sufficient to recover EKLF and GATA1 expression and differentiation can be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs are
competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement
of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B
receptor or of the inhibitory Smad6 protein prevents induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and
Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad
induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate
that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage (Adelman, 2002).
The transcription factor GATA-1 and its cofactor FOG-1 are essential for the normal development of erythroid cells and megakaryocytes. FOG-1 can stimulate or inhibit GATA-1 activity depending on cell and promoter context. How the GATA-1-FOG-1 complex controls the expression of distinct sets of gene in megakaryocytes and erythroid cells is not understood. The molecular basis for the megakaryocyte-restricted activation of the aIIb gene has been examined. FOG-1 stimulates GATA-1-dependent aIIb gene expression in a manner that requires their direct physical interaction. Transcriptional output by the GATA-1-FOG-1 complex is determined by the hematopoietic Ets protein Fli-1 that binds to an adjacent Ets element. Chromatin immunoprecipitation experiments show that GATA-1, FOG-1 and Fli-1 co-occupy the aIIb promoter in vivo. Expression of several additional megakaryocyte-specific genes that bear tandem GATA and Ets elements in their promoters also depends on the physical interaction between GATA-1 and FOG-1. These studies define a molecular context for transcriptional activation by GATA-1 and FOG-1, and may explain the occurrence of tandem GATA and Ets elements in the promoters of numerous megakaryocyte-expressed genes (Wang, 2002).
The developmental plasticity of transplanted adult stem cells challenges the notion that tissue-restricted stem cells have stringently limited lineage potential and prompts a re-evaluation of the stability of lineage commitment. Transformed cell systems are inappropriate for such studies, since transformation potentially dysregulates the processes governing lineage commitment. Therefore the stability of normal lineage commitment was assessed in primary adult hematopoietic cells. For these studies prospectively isolated primary bipotent progenitors were used; these normally display only neutrophil and monocyte differentiation in vitro.
GATA-1 was originally described as an erythroid lineage-affiliated transcription factor and has served as a paradigm for studies of tissue-specific transcription and lineage commitment. Cells were transduced with either a control retrovirus or a retrovirus containing a ligand-inducible form of GATA-1. In response to ectopic transcription factor expression, neutrophil/monocyte progenitors were reprogrammed to take on erythroid, eosinophil and basophil-like cell fates, with the resultant colonies resembling the mixed lineage colonies normally generated by multipotential progenitors. Clone-marking and daughter cell experiments identified lineage switching rather than differential cell selection as the mechanism of altered lineage output. These results demonstrate that the cell type-specific programming of apparently committed primary progenitors is not irrevocably fixed, but may be radically re-specified in response to a single transcriptional regulator (Hayworth, 2002).
The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. This study shows that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, the role of SCL is defined as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, it was demonstrated by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. It is therefore concluded that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, these observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells (Lécuyer, 2002).
Expression of Gfi (growth factor-independence)-1B (See Drosophila Senseless), a Gfi-1-related transcriptional repressor, is restricted to erythroid lineage cells and is essential for erythropoiesis. The transcription start site of the human Gfi-1B gene has been determined and its first non-coding exon has been located approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, it has been demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of the Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of the Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, these results have indicated that this GATA-1-mediated trans-activation is dependent on NF-Y binding to the CCAAT site. It is concluded that functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter (Huang, 2004)
Gata1 is a transcription factor essential for erythropoiesis. Erythroid cells lacking Gata1 undergo apoptosis, while overexpression of Gata1 results in a block in erythroid differentiation. However, erythroid cells overexpressing Gata1 differentiate normally in vivo when in the presence of wild-type cells. A model is proposed whereby a signal generated by wild-type cells (red cell differentiation signal; REDS) overcomes the intrinsic defect in Gata1-overexpressing erythroid cells. The simplest interpretation of this model is that wild-type erythroid cells generate REDS. To substantiate this notion, a tissue specific Cre/loxP system and the process of X-inactivation were exploited to generate mice that overexpress Gata1 in half the erythroid cells and are Gata1 null in the other half. The results show that the cells supplying REDS are erythroid cells. This study demonstrates the importance of intercellular signalling in regulating Gata1 activity and that this homotypic signalling between erythroid cells is crucial to normal differentiation (Gutiérrez, 2004).
GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, the three-dimensional structure of a complex comprising the interaction domains of these proteins has been determined. The structure reveals how zinc fingers can act as protein recognition motifs.
Notably, none of the FOG ZnFs that contact GATA-1 are part of tandem arrays of ZnFs. Thousands of such 'isolated' ZnFs exist, and it is likely that many serve as protein recognition motifs. The surface used by FOG ZnFs to recognize GATA-1 overlaps with the surface normally used by classical ZnFs to bind to DNA, indicating that the classical ZnF has acted throughout evolution as a versatile structural scaffold, onto which different binding functions have been 'grafted'. In line with this idea, the third classical ZnF from FOG has been shown to mediates a specific interaction with the coiled-coil protein TACC3. Indeed, given that a single classical ZnF is capable of mediating protein-protein interactions, and that an array of such domains is necessary for high affinity DNA binding, it is likely that the latter function arose later as a consequence of gene duplication events (Liew, 2004).
The Ski oncoprotein dramatically affects cell growth, differentiation, and/or survival. Recently, Ski was shown to act in distinct signaling pathways including those involving nuclear receptors, transforming growth factor β, and tumor suppressors. These divergent roles of Ski are probably dependent on Ski's capacity to bind multiple partners with disparate functions. In particular, Ski alters the growth and differentiation program of erythroid progenitor cells, leading to malignant leukemia. However, the mechanism underlying this important effect has remained elusive. This study shows that Ski interacts with GATA1, a transcription factor essential in erythropoiesis. Using a Ski mutant deficient in GATA1 binding, it was shown that this Ski-GATA1 interaction is critical for Ski's ability to repress GATA1-mediated transcription and block erythroid differentiation. Furthermore, the repression of GATA1-mediated transcription involves Ski's ability to block DNA binding of GATA1. This finding is in marked contrast to those in previous reports on the mechanism of repression by Ski, which have described a model involving the recruitment of corepressors into DNA-bound transcription complexes. It is proposed that Ski cooperates in the process of transformation in erythroid cells by interfering with GATA1 function, thereby contributing to erythroleukemia (Ueki, 2004).
GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. This study shows that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential antiapoptotic role during terminal erythroid differentiation. Loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late-stage erythroblasts. This programmed cell death is Arf and p53 independent and is instead mediated by upregulation of the proapoptotic factor Bim. Lrf was identified as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim loss delays the lethality of Lrf-deficient embryos and rescues their anemia phenotype. Thus, these data define a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF (Maeda, 2009).
Oncogene-mediated transformation of hematopoietic cells has been studied extensively, but little is known about the molecular basis for restriction of oncogenes to certain target cells and differential cellular context-specific requirements for oncogenic transformation between infant and adult leukemias. Understanding cell type-specific interplay of signaling pathways and oncogenes is essential for developing targeted cancer therapies. This study addresses the vexing issue of how developmental restriction is achieved in Down syndrome acute megakaryoblastic leukemia (DS-AMKL), characterized by the triad of fetal origin, mutated GATA1 (GATA1s), and trisomy 21. This study demonstrates overactivity of insulin-like growth factor (IGF) signaling in authentic human DS-AMKL and in a DS-AMKL mouse model generated through retroviral insertional mutagenesis. Fetal but not adult megakaryocytic progenitors are dependent on this pathway. GATA1 restricts IGF-mediated activation of the E2F transcription network to coordinate proliferation and differentiation. Failure of a direct GATA1-E2F interaction in mutated GATA1s converges with overactive IGF signaling to promote cellular transformation of DS fetal progenitors, revealing a complex, fetal stage-specific regulatory network. This study underscores context-dependent requirements during oncogenesis, and explains resistance to transformation of ostensibly similar adult progenitors (Klusmann, 2010).
Binding sites for the ubiquitously expressed transcription factor CP2 are present in regulatory regions of multiple erythroid genes. In these regions, the CP2 binding site was adjacent to a site for the erythroid factor GATA-1. Using three such regulatory regions (from genes encoding the transcription factors GATA-1, EKLF, and p45 NF-E2), the functional importance of the adjacent CP2/GATA-1 sites has been demonstrated. In particular, CP2 binds to the GATA-1 HS2 enhancer, generating a ternary complex with GATA-1 and DNA. Mutations in the CP2 consensus greatly impair HS2 activity in transient transfection assays with K562 cells. Similar results were obtained by transfection of EKLF and p45 NF-E2 mutant constructs. Chromatin immunoprecipitation with K562 cells showed that CP2 binds in vivo to all three regulatory elements and that both GATA-1 and CP2 are present on the same GATA-1 and EKLF regulatory elements. Adjacent CP2/GATA-1 sites may represent a novel module for erythroid expression of a number of genes. Additionally, coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated a physical interaction between GATA-1 and CP2. This may contribute to the functional cooperation between these factors and provide an explanation for the important role of ubiquitous CP2 in the regulation of erythroid genes (Bose, 2006; full text of article).
CP2 binds as a dimer to a CNRG (N5-6)CNRG DNA motif, present in diverse cellular and viral promoters. CP2c was originally identified by its ability to stimulate the transcription of the -globin gene; it appears to be involved in the fetal erythroid expression of the α-globin gene through the formation of a heterodimer with the erythroid transcription partner NF-E4 (the stage selector protein). Indeed, in transgenic experiments, the mutation of the stage selector protein binding site on the alpha-globin promoter (the stage selector element [SSE]) shows that this region affects the gamma- versus β-globin ratio of expression in early stages of fetal development. Moreover, overexpression of antisense CP2 mRNA in MEL cells undergoing erythroid differentiation in vitro not only suppresses α-globin expression but also impairs β-globin expression and hemoglobinization, suggesting that CP2 binding to the promoter is essential for optimal globin transcription in erythroid cells (Bose, 2006 and references therein).
The transcriptional regulation of GATA-1 is coordinated by several regulatory elements located both 5' to the transcriptional start site and in the first intron. The region upstream of the erythroid promoter, also reported as hypersensitivity site 2 (HS2), contains a double GATA motif shown to be essential for the erythroid promoter activity. This sequence, foot printed in vivo in erythroid cells, represents a strong double GATA-1 binding site, which has been proposed to mediate autoregulation by GATA-1 itself. Mice harboring a 21-base-pair deletion in this motif in the endogenous GATA-1 locus show a lack of eosinophil production, while platelets and mast cells appear normal. Erythropoiesis is also abnormal in these animals, which display a reduction in red cell number, hematocrit, and hemoglobin (Bose, 2006 and references therein).
The common role of GATA-1 and CP2 in regulating erythroid genes suggested the possibility that this could be occurring cooperatively. This study shows that this is true for GATA-1 gene transcription, in which the two factors act through adjacent binding sites present on the HS2 erythroid enhancer element. CP2 binds to HS2, creating a ternary complex with GATA-1 and DNA, and mutations in the CP2 consensus greatly impair HS2 activity in transient transfection assays with K562 cells. Adjacent GATA-1 and CP2 binding sites are also present in regulatory elements of several other genes expressed in the hematopoietic lineage. Among them, it is shown that a CP2 binding site adjacent to functionally relevant GATA-1 sites is important for the activities of the p45NF-E2 and EKLF promoters. Chromatin immunoprecipitation (ChIP) experiments reveal that CP2 is bound in vivo to the regulatory elements of the GATA-1, EKLF, and p45 NF-E2 genes and that at least for the first two genes, it is bound simultaneously to GATA-1. Finally, GATA-1 and CP2 can physically interact, even in the absence of DNA, as demonstrated by immunoprecipitation and glutathione S-transferase pull-down experiments. Taken together, these data suggest that adjacent GATA-1 and CP2 sites contribute to the regulation of the GATA-1, p45 NF-E2, and EKLF genes (Bose, 2006).
The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) have been shown to interact with Ldb1 and are also important in developmental programs. Two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins (Xu, 2007).
When the orphan nuclear receptors TR2 and TR4, the DNA-binding subunits of the DRED repressor complex, are forcibly expressed in erythroid cells of transgenic mice, embryos exhibit a transient mid-gestational anemia as a consequence of a reduction in the number of primitive erythroid cells. GATA-1 mRNA is specifically diminished in the erythroid cells of these TR2/TR4 transgenic embryos as it is in human CD34+ progenitor cells transfected with forcibly expressed TR2/TR4. In contrast, in loss-of-function studies analyzing either Tr2- or Tr4-germline-null mutant mice or human CD34+ progenitor cells transfected with force-expressed TR2 and TR4 short hairpin RNAs (shRNAs), GATA-1 mRNA is induced. An evolutionarily conserved direct repeat (DR) element, a canonical binding site for nuclear receptors, was identified in the GATA1 hematopoietic enhancer (G1HE), and TR2/TR4 binds to that site in vitro and in vivo. Mutation of that DR element led to elevated Gata1 promoter activity, and reduced promoter responsiveness to cotransfected TR2/TR4. Thus, TR2/TR4 directly represses Gata1/GATA1 transcription in murine and human erythroid progenitor cells through an evolutionarily conserved binding site within a well-characterized, tissue-specific Gata1 enhancer, thereby providing a mechanism by which Gata1 can be directly silenced during terminal erythroid maturation (Tanabe, 2007).
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