Regulation of SUP expression identifies multiple regulators involved in arabidopsis floral meristem development

During the course of flower development, floral homeotic genes are expressed in defined concentric regions of floral meristems called whorls. The SUPERMAN (SUP, also called FLO10) gene, which encodes a C2H2-type zinc finger protein, is involved in maintenance of the stamen/carpel whorl boundary (the boundary between whorl 3 and whorl 4) in Arabidopsis. Here, we show that the regulation of SUP expression in floral meristems is complex, consisting of two distinct phases, initiation and maintenance. The floral meristem identity gene LEAFY (LFY) plays a role in the initiation phase through at least two pathways, which differ from each other in the involvement of two homeotic genes, APETALA3 (AP3) and PISTILLATA (PI). AP3, PI, and another homeotic gene, AGAMOUS (AG), are further required for SUP expression in the later maintenance phase. Aside from these genes, there are other as yet unidentified genes that control both the temporal and spatial patterns of SUP expression in whorl 3 floral meristems. SUP appears to act transiently, probably functioning to trigger a genetic circuit that creates the correct position of the whorl 3/whorl 4 boundary.     

Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos

The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. We show that polyhomeotic(proximal) but not polyhomeotic(distal) is necessary for mitosis. To test if other PcG genes have roles in mitosis, we examined embryos derived from heterozygous PcG mutant females for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. We propose that these mitotic defects in PcG mutants delay cell cycle progression. We discuss possible mitotic roles for PcG proteins, and suggest that delays in cell cycle progression might lead to failure of maintenance.     

Stabilization of chromatin structure by PRC1, a Polycomb complex.

The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones.     

UNUSUAL FLORAL ORGANS Controls Meristem Identity and Organ Primordia Fate in Arabidopsis

A novel gene that is involved in regulating flower initiation and development has been identified in Arabidopsis. This gene has been designated UNUSUAL FLORAL ORGANS (UFO), with five corresponding nuclear recessive alleles designated ufo[middot]1 to ufo[middot]5. Under short day-length conditions, ufo homozygotes generate more coflorescences than do the wild type, and coflorescences often appear apical to the first floral shoot, resulting in a period of inflorescence development in which regions of floral and coflorescence shoots are produced alternately. ufo enhances the phenotype of weak leafy alleles, and the double mutant Ufo-1 Apetala1-1 produces only coflorescence-like shoots, suggesting that these two genes control different aspects of floral initiation. Floral development was also altered in Ufo plants. Ufo flowers have an altered organ number in all whorls, and organs in the first, second, and third whorls exhibit variable homeotic transformations. Ufo single and double mutant phenotypes suggest that the floral changes result from reduction in class B floral homeotic gene expression and fluctuations in the expression boundaries of class C function and FLO10. Surprisingly, in situ hybridization analysis revealed no obvious differences in expression pattern or level in developing Ufo flowers compared with that of the wild type for any class B or C gene studied. We propose that UFO acts in concert with known floral initiation genes and regulates the domains of floral homeotic gene function.     

A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression.

Segmentation genes provide the signals for the activation and regulation of homeotic genes in Drosophila but cannot maintain the resulting pattern of expression because their activity ceases halfway through embryogenesis. Maintenance of the pattern is due to the Polycomb group of genes (Pc-G) and the trithorax group of genes (trx-G), responsible for the persistence of the active or repressed state of homeotic genes. We have identified a regulatory element in the Ubx gene that responds to Pc-G and trx-G genes. Transposons carrying this element create new binding sites for Pc-G products in the polytene chromosomes. This Pc-G maintenance element (PRE), establishes a repressive complex that keeps enhancers repressed in cells in which they were originally repressed and maintains this state through many cell divisions. The trx-G products stimulate the expression of enhancers in cells in which they were originally active. This mechanism is responsible for the correct regulation of imaginal disc enhancers, which lack themselves antero-posterior positional information. The PRE also causes severe variegation of the mini-white gene present in the transposon, a phenomenon very similar to heterochromatic position-effect variegation. The significance of this mechanism for homeotic gene regulation is discussed.     

Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.

The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.     

Establishment of the Deformed expression stripe requires the combinatorial action of coordinate, gap and pair-rule proteins.

In Drosophila embryos, anterior-posterior positional identities are set and maintained by the expression boundaries of homeotic selector genes. The establishment of the initial expression boundaries of the homeotic genes are in turn dependent on earlier acting patterning genes of Drosophila. To define the combinations of early genes that are required to establish a unique blastoderm stripe of expression of the homeotic gene Deformed, we have analysed single and double patterning mutants and heat shock promoter fusion constructs that ectopically express early acting regulators. We find that the activation of Deformed is dependent on combinatorial input from at least three levels of the early hierarchy. The simplest activation code sufficient to establish Deformed expression, given the absence of negative regulators such as fushi-tarazu, consists of a moderate level of expression from the coordinate gene bicoid, in combination with expression from both the gap gene hunchback, and the pair-rule gene even-skipped. In addition, the activation code for Deformed is redundant; other pair-rule genes in addition to even-skipped can apparently act in combination with bicoid and hunchback to activate Deformed.     

Ectopic expression of a single homeotic gene, the Petunia gene green petal, is sufficient to convert sepals to petaloid organs.

Genetic studies in Arabidopsis and Antirrhinum showed that petal determination requires the concomitant expression of two homeotic functions, A and B, whereas the A function alone determines sepal identity. The B function is represented by at least two genes. The Petunia homeotic gene green petal (gp) is essential for petal determination as demonstrated by a Petunia gp mutant that has sepals instead of petals. We have used ectopic expression of the gp gene as a tool to study flower development in Petunia. CaMV 35S-gp expression leads to homeotic conversion of sepals into petaloid organs when expressed early in development. This demonstrates that a single homeotic gene is sufficient to induce homeotic conversion of sepals to petals, suggesting that other petal determining genes are regulated in part by ectopically expressed gp. Indeed, two other MADS-box-containing genes, pmads 2 and fbp 1, which show homology to the Antirrhinum B function gene globosa, are activated in the converted petal tissue. Furthermore, our data provide evidence for autoregulation of gp expression in the petaloid tissue and uncover the role of gp in fusion of petal tissues.     

Mutations in ccf, a novel Drosophila gene encoding a chromosomal factor, affect progression through mitosis and interact with Pc-G mutations.

We report herein the isolation of ccf, a new gene located in region 82E and essential for Drosophila development. This gene, expressed throughout development, encodes a novel product of 68 kDa which is found in the nucleus during interphase and labels, in a novel pattern, centrosomes and chromosome arms during mitosis. Mutations in ccf give rise to late larvae with small imaginal discs and to adults showing appendages of reduced size, consistent with CCF involvement in cell proliferation. Neuroblast squash analyses show that CCF is required for proper condensation of mitotic chromosomes and, therefore, for progression through mitosis. Furthermore, we observe that adult ccf mutants as well as animals overexpressing CCF during larval stages exhibit homeotic transformations. We also find that mutations in the Pc-G genes Polycomb, polyhomeotic and Enhancer of zeste are enhanced by ccf mutations. Finally, we show that the CCF protein binds to specific sites on polytene chromosomes, many of which are shared with the Posterior sex combs Pc-G protein. Together, these results suggest a role for the CCF protein in the maintenance of chromosome structure during mitosis and interphase.