Krüppel


EVOLUTIONARY HOMOLOGS

To obtain a clearer understanding of the evolutionary transition between short- and long-germ modes of embryogenesis in insects, the expression of two gap genes hunchback (hb) and Kruppel (Kr) as well as the pair-rule gene even-skipped (eve) were studied in the dipteran Clogmia albipunctata (Nematocera, Psychodidae). Embryogenesis in this species has features of both short- and long-germ modes of development. In Clogmia, hb expression deviates from that known in Drosophila in two main respects: (1) it shows an extended dorsal domain that is linked to the large serosa anlage, and (2) it shows a terminal expression in the proctodeal region. These expression patterns are reminiscent of the hb expression pattern in the beetle Tribolium, which has a short germ mode of embryogenesis (see Tribolium early embryonic development). However, Kruppel expression is rather similar to the Drosophila expression, both at early and late stages. eve expression starts with six stripes formed at blastoderm stage, while the seventh is only formed after the onset of gastrulation and germband extension. Surprisingly, no segmental secondary Eve stripes could be observed in Clogmia although such segmental stripes are known from higher dipterans, beetles and hymenopterans. Another nematoceran, Coboldia, was therefore studied to address this question and it was found that some segmental stripes form by intercalation as in Drosophila, although belatedly. These results suggest that Clogmia embryogenesis, both with respect to morphological and molecular characteristics represents an intermediate between the long-germ mode known from higher dipterans such as Drosophila, and the short-germ mode found in more ancestral insects (Rohr, 1999).

Krüppel is a gap gene in the intermediate germband insect Oncopeltus fasciatus and is required for development of both blastoderm and germband-derived segments

Segmentation in long germband insects such as Drosophila occurs essentially simultaneously across the entire body. A cascade of segmentation genes patterns the embryo along its anterior-posterior axis via subdivision of the blastoderm. This is in contrast to short and intermediate germband modes of segmentation where the anterior segments are formed during the blastoderm stage and the remaining posterior segments arise at later stages from a posterior growth zone. The biphasic character of segment generation in short and intermediate germ insects implies that different formative mechanisms may be operating in blastoderm-derived and germband-derived segments. In Drosophila, the gap gene Krüppel is required for proper formation of the central portion of the embryo. This domain of Krüppel activity in Drosophila corresponds to a region that in short and intermediate germband insects spans both blastoderm and germband-derived segments. The Krüppel homolog from the milkweed bug, Oncopeltus fasciatus (Hemiptera, Lygaeidae), an intermediate germband insect, has been cloned. Oncopeltus Krüppel is expressed in a gap-like domain in the thorax during the blastoderm and germband stages of embryogenesis. In order to investigate the function of Krüppel in Oncopeltus segmentation, knockdown phenotypes using RNAi were generated. Loss of Krüppel activity in Oncopeltus results in a large gap phenotype, with loss of the mesothoracic through fourth abdominal segments. Additionally, Krüppel is required to suppress both anterior and posterior Hox gene expression in the central portion of the germband. These results show that Krüppel is required for both blastoderm-derived and germband-derived segments and indicates that Krüppel function is largely conserved in Oncopeltus and Drosophila despite their divergent embryogenesis (Liu, 2004).

Three observations were made that may have implications for the evolution of insect segmentation. (1) Some of the molecular underpinnings may be shared between blastoderm-derived and germband-derived segments. However, in Tribolium, another short germ insect, the abdominal gap gene giant does not act as a canonical gap gene. Instead, Tribolium giant may have a more general role in segmentation, suggesting that some aspects of abdominal segmentation have diverged. Nevertheless, Oncopeltus Kr does act as a true gap gene, suggesting that at least some of the mechanisms underlying segment formation may be shared between the milkweed bug blastoderm and germband as well as with Drosophila (Liu, 2004).

(2) Kr RNAi embryos ectopically express Dfd in a posterior domain and abd-A in an anterior domain. Oncopeltus Kr is not only required for the formation of segments in the middle portion of the embryo, but also regulates anterior and posterior genes. Kr may directly regulate these Hox genes or instead, Kr may regulate other gap genes as is the case in Drosophila and these may in turn regulate the downstream Hox genes. Thus in Oncopeltus, Kr seems to act as a sort of 'spacer' both to specify central segments and to prevent central expression of anterior and posterior genes (Liu, 2004).

(3) Although loss of Krüppel function results in a deletion of anterior abdominal segments, posterior abdominal segments appear normal. Since all abdominal segments are normally produced through elongation of the posterior germband, presence of normal posterior abdominal segments in Kr RNAi embryos means that for these segments, the segment formation function of the growth zone was not disrupted. Although called a 'growth zone', this region has not yet been well studied in insects, and it has yet to be shown to share characteristics with the growth zones of other arthropods such as spiders (Liu, 2004).

Nevertheless, the results imply that the segment formation function by the growth zone can, to some degree, be decoupled from the actual number of segments that it produces (Liu, 2004).

The above observations suggest a possible (and admittedly speculative) mechanism for transition between short, intermediate and long germ forms of segmentation. For instance, the results show that alterations in activity of a gap gene can change the number of segments that are normally specified at the blastoderm stage. Evolutionarily, this can perhaps be accomplished by decreasing the width of the Krüppel domain on the blastoderm while maintaining the number of segments that it specifies. This would allow more posterior genes to be expressed on the blastoderm and would serve to pack more gap domains (and therefore body regions) on the blastoderm fate map. Although the number of segments the growth zone produces can increase or decrease, the segment formation ability of the growth zone seems to be largely independent, and the remainder of the posterior segments would be generated as usual, via germband growth. This would result in shifting the relative number of segments generated at the blastoderm stage versus the germband stage - in effect converting a shorter germ insect into a longer germ insect. The above scenario is highly speculative and no doubt overly simplistic but is attractive because it offers a mechanism for evolving the mode of segmentation. At this point, it is clear that further work needs to be done. Functional analysis of the segmentation genes in short germ insects has only begun but should provide a greater understanding of these questions and conundrums in insect segmentation (Liu, 2004).

Breakdown of abdominal patterning in the Tribolium Krüppel mutant jaws

During Drosophila segmentation, gap genes function as short-range gradients that determine the boundaries of pair-rule stripes. A classical example is Drosophila Krüppel (Dm'Kr) which is expressed in the middle of the syncytial blastoderm embryo. Patterning defects in Dm'Kr mutants are centred symmetrically around its bell-shaped expression profile. The role of Krüppel was examined in the short-germ beetle Tribolium castaneum where the pair-rule stripes corresponding to the 10 abdominal segments arise during growth stages subsequent to the blastoderm. The previously described mutation jaws is an amorphic Tc'Kr allele. Pair-rule gene expression in the blastoderm is affected neither in the amorphic mutant nor in Tc'Kr RNAi embryos. Only during subsequent growth of the germ band does pair-rule patterning become disrupted. However, only segments arising posterior to the Tc'Kr expression domain are affected, i.e., the deletion profile is asymmetric relative to the expression domain. Moreover, stripe formation does not recover in posterior abdominal segments, i.e., the Tc'Krjaws phenotype does not constitute a gap in segment formation but results from a breakdown of segmentation past the 5th eve stripe. Alteration of pair-rule gene expression in Tc'Krjaws mutants does not suggest a direct role of Tc'Kr in defining specific stripe boundaries as in Drosophila. Together, these findings show that the segmentation function of Krüppel in this short-germ insect is fundamentally different from its role in the long-germ embryo of Drosophila. The role of Tc'Kr in Hox gene regulation, however, is in better accordance to the Drosophila paradigm (Cerny, 2005).

The most obvious differences between the phenotypes of Krüppel in Tribolium and Drosophila are the homeotic transformations in Tc'Krjaws and Tc'Kr RNAi larvae that are not evident in Dm'Kr mutants. Such transformations are not entirely unexpected given that in Drosophila the expression boundaries of Hox genes are also set by gap genes, including Dm'Kr. However, in Drosophila gap mutants all segments that would be transformed because of misregulation of homeotic genes usually also suffer segmentation defects and fail to develop. By contrast, Tribolium segment primordia anterior of, and within, the Krüppel expression domain do differentiate, such that homeotic transformations can manifest themselves in the differentiated larva (Cerny, 2005).

The expression of homeotic genes in Tc'Krjaws embryos is consistent with the morphological transformations observed. The results with Tc'Dfd, Tc'Scr, Tc'Antp and Tc'Ubx confirm and extend earlier findings for Tc'pb and Tc'UBX/Tc'ABD-A expression. Notably, the complementary double-segmental expression of Dfd and Scr in Tc'Krjaws embryos explains the phenotype of alternating maxillary and labial segments. These expression patterns indicate that the posterior limit of Tc'Dfd and Tc'Scr domains is set through inhibition by Tc'Kr. In this respect, Tc'Kr fulfils a function similar to Drosophila gap genes (Cerny, 2005).

The homeotic phenotype of Tc'gt RNAi embryos could suggest a similar function in Hox regulation for Tc'gt. Indeed Tc'Antp anteriorly expands and gnathal Hox genes (Tc'Scr) repress in Tc'gt RNAi embryos, consistent with the expansion of thoracic fates found in differentiated Tc'gt RNAi larvae. These transformations are just opposite to those of Tc'Krjaws larvae. Interestingly, in embryos that lack Tc'Kr and at the same time have reduced Tc'gt activity, the homeotic effect of Tc'Krjaws clearly is epistatic. This shows that the ectopic Tc'gt stripes in the Tc'Kr mutant do not contribute to the Tc'Kr phenotype. However, this experiment suggests that the homeotic transformation of gnathal segments into thorax in Tc'gt RNAi embryos is indeed an indirect effect and comes about through misregulation of Tc'Kr in these embryos. This interpretation is supported by the finding that the Tc'Kr expression domain expands anteriorly in Tc'gt RNAi embryos. Evidently, it is expansion of Tc'Kr that results in repression of gnathal Hox genes in maxilla and labium of Tc'gt RNAi embryos, not loss of gnathal Hox gene activation. Similarly, expansion of Tc'Antp in Tc'gt RNAi larvae could be due to activation by anteriorly expanded Tc'Kr. However, as Antp is not significantly reduced in Tc'Krjaws, it seems more likely that Tc'gt acts directly to define the anterior boundary of the Tc'Antp domain (Cerny, 2005).

In addition to gap gene input, Drosophila Hox genes also receive input from pair-rule genes. The near-pair-rule pattern of Tc'Dfd and Tc'Scr in Tc'Krjaws embryos reveals an important role of pair-rule genes also in defining Tribolium Hox domain boundaries. It seems likely that regulation of Tc'Dfd and Tc'Scr by pair-rule genes is responsible for the precision of their expression boundaries in wild-type Tribolium embryos, while input from gap genes defines the broad region where a particular Hox gene can become active (Cerny, 2005).

In Drosophila, Krüppel is expressed in a bell-shaped profile centered over the primordia of segments T2 to A3. In the Tribolium blastoderm, only one such gradient is present; the Tc'Kr domain covers the posterior pole. When the germ rudiment has formed, the Tc'Kr domain retracts from the posterior end and forms a distinct domain overlapping the three thoracic segment primordia. At this stage, therefore, the Tc'Kr domain covers more anterior segment primordia (and more anterior pair-rule stripes) than does its Drosophila counterpart (Cerny, 2005).

Both boundaries of the Dm'Kr expression domain have been shown to serve as short-range gradients that provide positional information to define the margins of pair-rule stripes. A similar function should have been expected at least for the anterior boundary of Tc'Kr, which already forms during the syncytial blastoderm. However, although this anterior boundary evidently is used for limiting gnathal Hox gene expression, it is not required for the formation of gnathal or thoracic segments. The segment polarity genes Tc'en and Tc'wg are expressed normally in all segments up to the first abdominal segment. In addition, the first four pair-rule stripes of Tc'eve show little or no change compared with wild type. The same is the case for the first four stripes of Tc'hairy and Tc'runt. Severe deviations from the wild-type pattern only become apparent beginning with the 5th Tc'eve stripe. These data clearly show that the Krüppel domain in Tribolium has no significant role in generating those primordia that arise within the reach of its blastoderm expression domain (Cerny, 2005).

Shortly after germ rudiment formation, the growth zone becomes free of Tc'Kr transcript, and this newly arising posterior border of the Tc'Kr domain could, in principle, provide positional information to regulate posterior pair-rule stripes. It is also argued that the posterior boundary of the Tc'Kr domain is unlikely to function as a direct instructive gradient for pair-rule genes. In Drosophila, gap genes usually define pair-rule stripe boundaries through repression. Accordingly, in gap mutants the corresponding pair-rule stripes expand towards the region where the gap domain normally resides. No such expansion of Tc'eve (or Tc'runt or Tc'hairy) stripes is observed in Tc'Krjaws, however, arguing against the idea that the anterior boundaries of abdominal pair-rule stripes were directly specified by Tc'Kr. Because at least one posterior segmentation gene domain (the abdominal domain of Tc'hb) does expand anteriorly in Tc'Krjaws, the lack of expanding pair-rule stripes is probably not due to the particular situation of the growing germ band but indeed reflects a genuine difference in the way that pair-rule stripes depend on Krüppel function in Tribolium versus Drosophila (Cerny, 2005).

Compared with the classical gap phenotype of Dm'Kr mutants, the segmental defects in Tc'Krjaws are shifted toward posterior. Based on its larval phenotype, Tc'Krjaws has been described as a gap gene, in that most abdominal segments are deleted while gnathal and thoracic segments, as well as the most posterior abdominal segments (A9 and A10), remain intact. However, when analysing pair-rule and segment-polarity expression, no observe resumption of stripe formation was observed posterior of a defect zone, as is observed for the mutation krusty, for example The progression of the en/wg pattern in Tc'Krjaws embryos is interpreted as reflecting a breakdown of segmentation, not a temporal gap in the sequence of abdominal segment additions. While the 9th and 10th abdominal segments usually are present in Tc'Krjaws mutant and Tc'Kr RNAi larvae and give rise to urogomphi and pygopods, it is concluded from a time series that these structures actually derive from the fragmentary stripes formed immediately after the anterior seven unaffected stripes have been generated. This implies that the remnants of middle-abdominal segments later on differentiate as posterior abdominal segments in Tc'Krjaws mutant embryos. To explain the specification of earlier formed segments as A9 and A10, it is speculated that after completion of germ band growth, a signal emanates from the posterior terminalia and instructs the next two segments to fuse with the telson and to form urogomphi and pygopods. In addition, non-segmental terminal structures are present in Tc'Krjaws embryos. These primordia are known to arise early in the blastoderm, posterior of the growth zone proper. One marker for terminal structures is the posterior terminal domain of Tc'wg, which is formed and maintained in Tc'Krjaws embryos similar to wild type. In addition, the cuticle lining of the hindgut is present in mutant larvae (Cerny, 2005).

Since the growth zone is a patterning environment very different from the syncytial blastoderm, it was expected that segmentation genes in short germ embryos would play similar roles as in Drosophila during early stages, while abdominal segmentation has been predicted to be fundamentally different. It is surprising that knock-down of several short germ gap gene homologues, i.e., Tc'gt, Tc'Kr, Gb'hb and Of'hb, results mainly in homeotic transformations in those segments that form during the blastoderm. This also pertains to Tc'hb, where homeotic transformations occur in addition to segmentation defects. That so many of these gap gene homologues do not seem to have strong roles in the formation of anterior segments raises the possibility that the original role of gap genes early during arthropod evolution may have been to regulate Hox genes, but not to directly regulate pair-rule genes. In Tribolium, however, some blastoderm pair-rule stripes are affected by gap gene orthologues other than Kr, and there is good evidence for stripe-specific elements driving at least the first two Tc'hairy stripes (Cerny, 2005).

The results for Tc'Kr deviate from those obtained for Krüppel in Oncopeltus fasciatus. In this short-germ insect, knock-down of Kr also results in mis-expression of Hox genes, although the effects are more limited as only one ectopic Of'Dfd domain is detected. Interestingly, expression of Of'en in such embryos seems to indicate a clear gap phenotype, i.e. perfect segmental stripes reappear posterior to a region of segmental disruption. Incomplete inactivation of Of'Kr could be responsible for this difference; it is noted, however, that weak Tc'Kr RNAi situations do not result in obvious gap phenotypes. Rather, in such embryos the segmentation process simply breaks down somewhat later than in Tc'Krjaws, i.e., the additional segments present in weak Tc'Kr RNAi embryos appear to represent anterior abdominal rather than posterior (post-gap) abdominal segments. Oncopeltus is sometimes denoted an intermediate-germ insect, because a few more segments are formed already in the blastoderm than, for example, in Tribolium. It will be interesting to see if the 'next posterior' gap gene in Oncopeltus will also display a 'gap' phenotype, and to find out whether pair-rule gene expression in Of'Kr RNAi embryos indicates a role in the regulation of specific stripes boundaries (Cerny, 2005).

If the interpretation is correct that Tc'Kr does not directly specify pair-rule stripes during abdomen formation, what could its function be in this process? All abdominal cells derive from progenitors that expressed Tc'Kr at the blastoderm stage. Therefore, regulation of later-acting abdominal expression domains (e.g., the posterior domains of Tc'gt and Tc'hb), may depend on Tc'Kr activity in the blastoderm, rather than on its activity at later stages when its domain forms a distinct posterior boundary. In this way, the long-ranging action of Tc'Kr could be explained through a temporal persistence rather than a spatial diffusion mechanism. Later acting genes depending on Tc'Kr activity then could have a role in regulating pair-rule genes (Cerny, 2005).

However, the discovery that a segmentation clock appears to pattern lower arthropods raises the issue of when in the evolutionary line leading to the diptera this clock was replaced by the hierarchical mode of Drosophila segmentation. Although at present no evidence is available for a segmentation clock functioning in Tribolium, it is conceivable that a modified clock is installed at the posterior end of the blastoderm embryo. Tc'Kr could have a role in initiation of this clock machinery. Alternatively, it could be required for its continued function. Because the number of abdominal segments is constant in insects, some type of counting principle would be required to stop the clock once the last segment has formed. Such a counting mechanism could be provided, for example, by a series of abdominal 'gap gene' activities (including the posterior domains of Tc'gt and Tc'hb), the last of which would shut off the clock. In this view, abdominal 'gap genes' would have a permissive rather than a positionally instructive function during abdominal segmentation of short germ embryos (Cerny, 2005).

The gap gene Kruppel of Rhodnius prolixus is required for segmentation and for repression of the homeotic gene sex comb-reduced

The establishment of the anterior-posterior segmentation in insects requires the concerted action of a hierarchical gene network. The orthologue of Kruppel gap gene was studied in the hemipteran Rhodnius prolixus (Rp-Kr). Its structure, expression pattern and function were characterized. The genomic sequence upstream of the Rp-Kr transcriptional unit shows a putative regulatory region conserved in the orthologue genes from Drosophila melanogaster and Tribolium castaneum. Rp-Kr expression is zygotic and it is expressed in the anterior half of the embryo (the posterior half of the egg) during the blastoderm stage and germ band formation; later, during germ band extension, it is expressed in a central domain, from T2 to A3. The Rp-Kr loss of function phenotypes shows disrupted thoracic and abdominal segmentation. Embryos with weak segmentation phenotypes show homeotic transformations, in which an ectopic tibial comb, typical of T1 leg, appears in T2, which correlates with the ectopic expression of Rp-sex-comb reduced in this leg. It is concluded that Kruppel of Rhodnius prolixus is required for segmentation and for repression of the homeotic gene sex comb-reduced (Lavore, 2014).

Krüppel-like is required for nonskeletogenic mesoderm specification in the sea urchin embryo

The canonical Wnt pathway plays a central role in specifying vegetal cell fate in sea urchin embryos. SpKrl has been cloned as a direct target of nuclear β-catenin. Using Hemicentrotus pulcherrimus embryos, this study shows that HpKrl controls the specification of secondary mesenchyme cells (SMCs) through both cell-autonomous and non-autonomous means. Like SpKrl, HpKrl was activated in both micromere and macromere progenies. To examine the functions of HpKrl in each blastomere, chimeric embryos were constructed composed of blastomeres from control and morpholino-mediated HpKrl-knockdown embryos, and the phenotypes of the chimeras were analyzed. Micromere-swapping experiments showed that HpKrl is not involved in micromere specification, while micromere-deprivation assays indicated that macromeres require HpKrl for cell-autonomous specification. Transplantation of normal micromeres into a micromere-less host with morpholino revealed that macromeres are able to receive at least some micromere signals regardless of HpKrl function. From these observations, it is proposed that two distinct pathways of endomesoderm formation exist in macromeres, a Krl-dependent pathway and a Krl-independent pathway. The Krl-independent pathway may correspond to the Delta/Notch signaling pathway via GataE and Gcm. It is suggested that Krl may be a downstream component of nuclearβ-catenin required by macromeres for formation of more vegetal tissues, not as a member of the Delta/Notch pathway, but as a parallel effector of the signaling (Krl-dependent pathway) (Yamazaki, 2008).

Maternal activation of gap genes in the hover fly Episyrphus

The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, loss- and gain-of-function experiments were used in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. It was found that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. It is conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha) (Lemke, 2010).

Therefore, Episyrphus and other lower cyclorrhaphan flies establish global AP polarity only through bicoid and lack sizable input of nanos, although endogenous nanos activity in these species might stabilize the AP axis by repressing anterior development. Despite the absence of a redundant maternal system to generate global AP polarity, Eba-bcd appears to be a less potent transcriptional activator than Bicoid. In contrast to Drosophila, gap gene activation at the anterior pole of the Episyrphus embryo requires a strong contribution of the terminal system, whereas the posterior domains of knirps and giant are strictly dependent on caudal and do not appear to receive a significant activating input by Eba-bcd. Thus, rather than a strong activation potential, the exclusive control of the central Eba-Kr domain by Eba-bcd appears to be the crucial difference to Drosophila, which renders AP polarity in the Episyrphus embryo entirely dependent on bicoid (Lemke, 2010).

Segmentation gene expression patterns in Bactrocera dorsalis and related insects: regulation and shape of blastoderm and larval cuticle

The oriental fruit fly, Bactrocera dorsalis, is regarded as a severe pest of fruit production in Asia. Despite its economic importance, only limited information regarding the molecular and developmental biology of this insect is known to date. This study provides a detailed analysis of B. dorsalis embryology, as well as the expression patterns of a number of segmentation genes known to act during patterning of Drosophila and compare these to the patterns of other insect families. An anterior shift of the expression of gap genes was detected when compared to Drosophila. This shift was largely restored during the step where the gap genes control expression of the pair-rule genes. The shapes were analyzed of the embryos of insects of different families, B. dorsalis and the blow fly Lucilia sericata and compared with that of the well-characterized Drosophila melanogaster. Distinct shapes were found as well as differences in the ratios of the length of the anterior-posterior axis and the dorsal-ventral axis. These features were integrated into a profile of how the expression patterns of the gap gene Kruppel and the pair-rule gene even-skipped were observed along the A-P axis in three insects families. Since significant differences were observed, how Kruppel controls the even-skipped stripes is discussed. Furthermore, how the position and angles of the segmentation gene stripes differed from other insects is discussed. Finally, the outcome was analyzed of the expression patterns of the late acting segment polarity genes in relation to the anlagen of the naked-cuticle and denticle belt area of the B. dorsalis larva (Suksuwan, 2017).

Members of the EKLF multi-gene family

EKLF zinc fingers are most homologous to the Krüppel family of transcription factors. Because of the complexity of the zinc finger family of transcription factors, it cannot be asserted, however, that Krüppel and EKLF are true structural or functional homologs. Information about the EKLF family is provided for general interest only.

EKLF [erythroid Krüppel-like factor] was isolated by enriching for genes expressed in a mouse erythroleukemia cell line but not expressed in a mouse monocyte-macrophage cell line. The complete cDNA sequence is predicted to encode a protein of approximately 38,000 Da that contains a proline-rich amino domain and three TFIIIA-like zinc fingers within the carboxy domain. EKLF is able to bind the sequence CCA CAC CCT, an essential element of the beta-globin promoter. Its tissue distribution establishes that the EKLF transcript is expressed only in bone marrow and spleen, the two hematopoietic organs of the mouse (Miller, 1993).

The gut-enriched Kruppel-like factor (GKLF) is a newly identified transcription factor that contains three C2H2 Kruppel-type zinc fingers. Previous immunocytochemical studies indicate that GKLF is exclusively localized to the nucleus. To identify the nuclear localization signal (NLS) within GKLF, cDNA constructs with various deletions in the coding region of GKLF were generated and analyzed by indirect immunofluorescence in transfected COS-1 cells. In addition, constructs fusing regions representing putative NLSs of GKLF to green fluorescent protein (GFP) were generated and examined by fluorescence microscopy in similarly transfected cells. The results indicate that GKLF contains two potent, independent NLSs: one within the zinc fingers and the other in a cluster of basic amino acids (called the 5' basic region) immediately preceding the first zinc finger. In comparison, putative NLSs within the zinc fingers and the 5' basic region of a related Kruppel protein, zif268/Egr-1 (See Drosophila Stripe and Huckebein), are relatively less efficient in their ability to translocate GFP into the nucleus. A search in the protein sequence data base reveals that despite the existence of numerous Kruppel proteins, only two, the lung Kruppel-like factor (LKLF) and the erythroid Kruppel-like factor (EKLF), exhibit similar NLSs to those of GKLF. These findings indicate that GKLF, LKLF, and EKLF are members of a subfamily of closely related Kruppel proteins. These proteins are distantly related to Sp1 and even more distantly related to Drosophila Kruppel, which is only 45% similar in zinc fingers 2, 3 and 4 (Shields, 1997).

CACCC boxes are among the critical sequences present in regulatory elements of genes expressed in erythroid cells, as well as in other selected cell types. While an erythroid cell-specific CACCC-box-binding protein, EKLF is 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. Additional CACCC-box transcription factors were sought that might be active in murine erythroid cells. A major gel shift activity (termed BKLF), present in yolk sac and fetal liver erythroid cells, has been found that can be distinguished from EKLF by specific antisera. BKLF is a highly basic novel zinc finger protein that is related to EKLF and other Kruppel-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 (Crossley, 1996).

A novel zinc finger protein has been identified and named ubiquitous Kruppel-like factor (UKLF) based on structural considerations and the pattern of gene expression. UKLF was isolated by the polymerase chain reaction approach using degenerate oligonucleotides corresponding to the DNA-binding domain of erythroid Kruppel-like factor (EKLF) and cDNA prepared from human vascular endothelial cells. The carboxyl-terminal portion of UKLF contains three zinc fingers of the Cys2-His2 type and binds in vitro to the CACCC motif of the beta-globin promoter and to the Sp1 recognition sequence. The amino-terminal portion of UKLF consists of a hydrophobic region rich in serines and a negatively charged segment with several glutamic acid residues. The first 47 amino acids of the acidic region are nearly identical to the amino-terminal portion of another Kruppel-like factor, the so-called core promoter-binding protein (CPBP) or Zf9. Like CPBP/Zf9, UKLF can function as a transcription activator in co-transfection assays. However, this activity is lost when the highly conserved amino-terminal segment is deleted. These findings indicate that UKLF and CPBP/Zf9 represent a distinct subgroup of closely related Kruppel-like activators of transcription. Mapping of the UKLF gene to chromosome 2 suggests that UKLF and CPBP/Zf9 translocated to different chromosomes following duplication from an ancestral gene (Matsumoto, 1998).

The gut-enriched Kruppel-like factor (GKLF) is a recently identified eukaryotic transcription factor that contains three C2H2 zinc fingers. The amino acid sequence of the zinc finger portion of GKLF is closely related to several Kruppel proteins, including the lung Kruppel-like factor (LKLF), the erythroid Kruppel-like factor (EKLF) and the basic transcription element binding protein 2 (BTEB2). The DNA sequence to which GKLF binds has not been definitively established. The DNA binding sequence of GKLF was determined using highly purified recombinant GKLF in a target detection assay of an oligonucleotide library consisting of random sequences. Upon repeated rounds of selection and subsequent characterization of the selected sequences by base-specific mutagenesis, a DNA with the sequence 5'-G/AG/AGGC/TGC/T-3' was found to contain the minimal essential binding site for GKLF. This sequence is present in the promoters of two previously characterized genes: the CACCC element of the beta-globin gene, which interacts with EKLF, and the basic transcription element (BTE) of the CYP1A1 gene, which interacts with Sp1 and several Sp1-like transcription factors. Moreover, the selected GKLF binding sequence is capable of mediating transactivation of a linked reporter gene by GKLF in co-transfection experiments. These results establish GKLF as a sequence-specific transcription factor likely involved in the regulation of expression of endogenous genes (Shields, 1998).

Members of the erythroid Kruppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene, as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, a cDNA encoding a member of this family was cloned from a human vascular endothelial cell cDNA library. Sequence analysis indicates that the clone, hEZF, is the human homolog of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein binds specifically to a probe containing the CACCC core sequence. Sense hEZF (but not antisense hEZF) decreases by 6-fold the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter. In contrast, EKLF increases the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, a repression domain in hEZF was mapped to amino acids 181-388. Amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain (Yet, 1998).

Mutation of EKLF family members

Heterozygous EKLF+/- knockout mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- knockout mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin (Nuez, 1995).

EKLF targeting of the globin loci

Disruption of the gene for transcription factor EKLF (erythroid Krüppel-like factor) results in fatal anemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic beta- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the beta globin gene promoter, even though it is already present in embryonic red cells. A beta-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages; this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, the expression of LCR-driven lacZ reporters was analyzed. Results indicate that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation (Tewari, 1998).

The locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. The function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3. mSp1 is shown not act as an activator in this context. It is concluded that direct binding of EKLF is implicated in chromatin remodeling over 5'HS3, the only locus control region fragment that appears to have an intrinsic dominant chromatin opening activity (Gillemans, 1998).

Erythroid Kruppel-like factor (EKLF) is necessary for stage-specific expression of the human beta-globin gene. EKLF has been shown to require a SWI/SNF-related chromatin remodeling complex, EKLF coactivator-remodeling complex 1 (E-RC1), to generate a DNase I hypersensitive, transcriptionally active beta-globin promoter on chromatin templates in vitro. To examine the nucleosomal structure of in vitro-assembled ß-globin promoters, indirect end-labeling analysis of DNase I digested chromatin was performed. Importantly, these digestions were carried out prior to transcription and thus reflect potentially active, rather than transcribing, promoters. A DNase I hypersensitive region from approximately -120 to +10 in the beta-globin promoter is observed in the presence of both EKLF and E-RC1. This pattern resembles the hypersensitive structure of the active beta-globin promoter in vivo, which maps from 100-150 bp 5' of the cap site. Neither EKLF nor E-RC1 are sufficient alone to generate an open chromatin configuration. Since most chromatin remodeling events are energy-dependent, it was asked whether hypersensitive site formation in the beta-globin promoter is dependent on ATP. Chromatin templates were incubated with the ATP-hydrolyzing reagent apyrase prior to addition of EKLF and E-RC1. Indeed, formation of the open beta-globin promoter structure by these proteins requires ATP. E-RC1 contains BRG1, BAF170, BAF155, and INI1 (BAF47), all homologs of yeast SWI/SNF subunits, as well as a subunit unique to higher eukaryotes, BAF57, which is critical for chromatin remodeling and transcription with EKLF. E-RC1 displays functional selectivity toward transcription factors, since it cannot activate expression of chromatin-assembled HIV-1 templates with the E box-binding protein TFE-3. Thus, a member of the SWI/SNF family acts directly in transcriptional activation and may regulate subsets of genes by selectively interacting with specific DNA-binding proteins (Armstrong, 1998).

Erythroid Kruppel-like factor (EKLF) is a red cell-specific transcriptional activator that is crucial for consolidating the switch to high levels of adult beta-globin expression during erythroid ontogeny. EKLF is required for integrity of the chromatin structure at the beta-like globin locus, and it interacts with a positive-acting factor in vivo. EKLF is an acetylated transcription factor, and it interacts in vivo with CBP, p300, and P/CAF. However, its interactions with these histone acetyltransferases are not equivalent, since CBP and p300, but not P/CAF, utilize EKLF as a substrate for in vitro acetylation within EKLF's trans-activation regions. The functional effects of these interactions are that CBP and p300, but not P/CAF, enhance EKLF's transcriptional activation of the beta-globin promoter in erythroid cells. These results establish EKLF as a tissue-specific transcription factor that undergoes post-translational acetylation and suggest a mechanism by which EKLF is able to alter chromatin structure and induce beta-globin expression within the beta-like globin cluster (Zhang, 1998).

The SWI/SNF family of chromatin-remodeling complexes plays a key role in facilitating the binding of specific transcription factors to nucleosomal DNA in diverse organisms from yeast to man. Yet the process by which SWI/SNF and other chromatin-remodeling complexes activate specific subsets of genes is poorly understood. Mammalian SWI/SNF regulates transcription from chromatin-assembled genes in a factor-specific manner in vitro. The DNA-binding domains (DBDs) of several zinc finger proteins, including EKLF, interact directly with SWI/SNF to generate DNase I hypersensitivity within the chromatin-assembled ß-globin promoter. Interestingly, two SWI/SNF subunits (BRG1 and BAF155) are necessary and sufficient for targeted chromatin remodeling and transcriptional activation by EKLF in vitro. Remodeling is achieved with only the BRG1-BAF155 minimal complex and the EKLF zinc finger DBD, whereas transcription requires, in addition, an activation domain. In contrast, the BRG1-BAF155 complex does not interact or function with two unrelated transcription factors, TFE3 and NF-kappaB. It is concluded that specific domains of certain transcription factors differentially target SWI/SNF complexes to chromatin in a gene-selective manner and that individual SWI/SNF subunits play unique roles in transcription factor-directed nucleosome remodeling (Kadam, 2000).

The erythroid cell-specific transcription factor erythroid Krüppel-like factor (EKLF) is an important activator of ß-globin gene expression. EKLF achieves this by binding to the CACCC element at the ß-globin promoter via its zinc finger domain. The coactivators CBP and P300 interact with acetylate, and enhance its activity, helping to explain ELKF's role as a transcription activator. EKLF can also interact with the corepressors mSin3A and HDAC1 (histone deacetylase 1) through its zinc finger domain. When linked to a GAL4 DNA binding domain, full-length EKLF or its zinc finger domain alone can repress transcription in vivo. This repressive activity can be relieved by the HDAC inhibitor trichostatin A. Although recruitment of EKLF to a promoter is required to show repression, its zinc finger domain cannot bind directly to DNA and repress transcription simultaneously. In addition, the target promoter configuration is important for enabling EKLF to exhibit any repressive activity. These results suggest that EKLF may function in vivo as a transcription repressor and play a previously unsuspected additional role in regulating erythroid gene expression and differentiation (Chen, 2001).

The mechanism of definitive-stage epsilon-globin transcriptional inactivity was explored within a human beta-globin YAC expressed in transgenic mice. Focus was placed on the globin CAC and CAAT promoter motifs, as studies have indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid krüppel-like factor (EKLF) was placed in the epsilon-globin promoter at a position corresponding to that in the adult beta-globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation leads to EKLF-independent epsilon-globin transcription during definitive erythropoiesis. A second 4-bp substitution in the epsilon-globin CAAT sequence, which simultaneously disrupts a second DR element, further enhances ectopic definitive erythroid activation of epsilon-globin transcription, which surprisingly becomes EKLF dependent. Factors in nuclear extracts prepared from embryonic or adult erythroid cells that bind these elements in vitro were examined, a novel DR-binding protein (DRED) was identified whose properties are consistent with those expected for a definitive-stage epsilon-globin repressor. It is concluded that the suppression of epsilon-globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the epsilon-globin gene (Tanimoto, 2000).

In summary, the wild-type epsilon-globin gene promoter bears two intact DR sequences that can potentially bind to COUP-TFII in primitive erythroid cells. However, these sites are not functional as silencer elements at the primitive stage. Hence, EKLF binding (to the natural distal CAC site) and other proteins activate this promoter and confer EKLF-dependent expression to this gene. In definitive erythroid cells, a putative repressor protein, DRED, can bind to the DR sequences and interfere with EKLF binding. Consequently, the epsilon-globin gene is silenced in definitive-stage cells. The beta-globin gene promoter, in contrast, does not bear DR sites and is therefore not subject to silencing by any definitive stage-specific repressor. It is therefore apparent that the context of specific transcription factor binding sites in their promoters determine the stage-specific expression of the epsilon- and beta-globin genes. In the epsilon-globin promoter, this process normally requires the participation of both positive and negative factors (Tanimoto, 2000).

Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the {beta}-globin locus. When actively expressed, the cis-regulatory elements of the ß-globin locus are in proximity in the nuclear space, forming a compartment termed the Active Chromatin Hub (ACH). However, it is unknown which proteins are involved in ACH formation. EKLF, an erythroid transcription factor required for adult ß-globin gene transcription, is also required for ACH formation. It is concluded that transcription factors can play an essential role in the three-dimensional organization of gene loci (Drissen, 2004).

Chromatin conformation capture (3C) technology was sued to investigate the three-dimensional conformation of the mouse ß-globin locus in the absence of EKLF. Cells from E12.5 EKLF-/- and wild-type fetal livers were cross-linked with formaldehyde, followed by restriction enzyme digestion of the DNA. The samples were ligated under conditions that favor the ligation of DNA fragments that are physically connected through the cross-links. Quantitative PCR across the junctions is used to determine the relative cross-linking frequencies between restriction fragments in the locus. This provides an indication of the nuclear proximity of DNA fragments in vivo. Cross-linking frequencies were determined for a total of 66 junctions that can be formed between 12 selected HindIII fragments spread over ~170 kb of DNA encompassing the ß-globin gene cluster. The brain served as a nonexpressing control tissue, in which the ß-globin locus appears to adopt a linear conformation. In wild-type E12.5 fetal liver cells, high cross-linking frequencies are found with the LCR and 5'HS-62, indicating their proximity to the ßmaj promoter in vivo. In the absence of EKLF however, these cross-linking frequencies are much lower and no interaction with a distal site stands out clearly, showing that the ßmaj promoter does not participate stably in a spatial clustering of chromatin. A comparable pattern is observed with locus-wide cross-linking frequencies of a fragment containing 5'HS2. Together with 5'HS3, 5'HS2 is the most prominent transcriptional activating element of the LCR. Interactions with 5'HS-62, ßmaj, 3'HS1, and the other HS of the LCR are strongly reduced in the absence of EKLF, indicating that 5'HS2 requires the presence of EKLF to participate in the ACH (Drissen, 2004).

The results demonstrate that the complete ACH is not formed in the absence of EKLF. However, the observed cross-linking frequencies in EKLF-/- fetal liver cells are still higher than those found in nonexpressing brain cells, indicating a different, nonlinear, structure. To investigate this, the locus-wide cross-linking frequencies of restriction fragments containing 5'HS-62 and 5'HS4/5 were compared. These sites participate in the chromatin hub present in erythroid progenitor cells before the globin genes are transcribed. In wild-type fetal liver cells, 5'HS-62 is in proximity to the LCR, ßmaj and 3'HS1. In EKLF-/- fetal liver cells, 5'HS-62 interactions with the HS at the 5' side of the LCR and with the distal 3'HS1 still stand out, whereas all other cross-linking frequencies are strongly reduced. This indicates the presence of a globin chromatin hub, containing 5'HS-62/-60, the HS at the 5' side of the LCR, and 3'HS1. The same structure is apparent when analyzing locus-wide cross-linking frequencies of a restriction fragment containing 5'HS4/5 at the 5' side of the LCR (Drissen, 2004).

Transcriptional regulation of EKLF

Erythroid Kruppel-like factor (EKLF) is a zinc finger transcription factor required for beta-globin gene expression and is implicated as one of the key factors necessary for the fetal to adult switch in globin gene expression. In an effort to identify factors involved in the expression of this important erythroid-specific regulatory protein, the mouse EKLF gene has been isolated and the promoter region has been systematically analyzed. Initially, a reporter construct with 1150 base pairs of the EKLF 5'-region was introduced into transgenic mice and shown to direct erythroid-specific expression. The expression studies in erythroid cells were continued and a sequence element was identified consisting of two GATA sites flanking an E box motif. The three sites act in concert to elevate the transcriptional activity of the EKLF promoter. Each site is essential for EKLF expression indicating that the three binding sites do not work additively, but rather function as a unit. GATA-1 binds to the two GATA sites and evidence suggests the binding of another factor from erythroid cell nuclear extracts to the E box motif. These results are consistent with the formation of a quaternary complex composed of an E box dimer and two GATA-1 proteins binding at a combined GATA-E box-GATA activator element in the distal EKLF promoter (Anderson, 1998).

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 hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However, the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. This study shows how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis, as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver, Eklf expression is shown to be initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies, it was found that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population, followed by a switch to Gata1-controlled regulation of Eklf transcription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis (Lohmann, 2008).

Co-repressors of EKLF family members

Basic Kruppel-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. BKLF is capable of repressing transcription, and its repression domain has been mapped to the N-terminus. A two-hybrid screen against BKLF was carried out, and a novel clone was isolated encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein that binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. 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. 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 (Turner, 1998).

Interaction of EKLF family members with Brm factors

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).


Krüppel: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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