Genome-wide oscillations in G + C density and sequence conservation.

Eukaryotic genomes typically show a uniform G + C content among chromosomes, but on smaller scales, many species have a G + C density that fluctuates with a characteristic wavelength. This oscillation is evident in many insect species, with wavelengths ranging between 700 bp and 4 kb. Measures of evolutionary conservation oscillate in phase with G + C content, with conserved regions having higher G + C. Loci with large regulatory regions show more regular oscillations; coding sequences and heterochromatic regions show little or no oscillation. There is little oscillation in vertebrate genomes in regions with densely distributed mobile repetitive elements. However, species with few repeats show oscillation in both G + C density and sequence conservation. These oscillations may reflect optimal spacing of cis-regulatory elements.

Molecular Lessons from the Drosophila Bithorax Complex.

The Genetics Society of America's (GSA's) Edward Novitski Prize recognizes a single experimental accomplishment or a body of work in which an exceptional level of creativity, and intellectual ingenuity, has been used to design and execute scientific experiments to solve a difficult problem in genetics. The 2020 recipient is Welcome W. Bender of Harvard Medical School, recognizing his creativity and ingenuity in revealing the molecular nature and regulation of the bithorax gene complex.

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.

Noncoding RNAs of the Ultrabithorax domain of the Drosophila bithorax complex.

RNA transcripts without obvious coding potential are widespread in many creatures, including the fruit fly, Drosophila melanogaster. Several noncoding RNAs have been identified within the Drosophila bithorax complex. These first appear in blastoderm stage embryos, and their expression patterns indicate that they are transcribed only from active domains of the bithorax complex. It has been suggested that these noncoding RNAs have a role in establishing active domains, perhaps by setting the state of Polycomb Response Elements A comprehensive survey across the proximal half of the bithorax complex has now revealed nine distinct noncoding RNA transcripts, including four within the Ultrabithorax transcription unit. At the blastoderm stage, the noncoding transcripts collectively span ∼75% of the 135 kb surveyed. Recombination-mediated cassette exchange was used to invert the promoter of one of the noncoding RNAs, a 23-kb transcript from the bxd domain of the bithorax complex. The resulting animals fail to make the normal bxd noncoding RNA and show no transcription across the bxd Polycomb Response Element in early embryos. The mutant flies look normal; the regulation of the bxd domain appears unaffected. Thus, the bxd noncoding RNA has no apparent function.

The border between the ultrabithorax and abdominal-A regulatory domains in the Drosophila bithorax complex.

The bithorax complex in Drosophila melanogaster includes three homeobox-containing genes--Ultrabithorax (Ubx), abdominal--A (abd-A), and Abdominal-B (Abd-B)-which are required for the proper differentiation of the posterior 10 segments of the body. Each of these genes has multiple distinct regulatory regions; there is one for each segmental unit of the body plan where the genes are expressed. One additional protein- coding gene in the bithorax complex, Glut3, a sugar-transporter homolog, can be deleted without phenotype. We focus here on the upstream regulatory region for Ubx, the bithoraxoid (bxd) domain, and its border with the adjacent infraabdominal-2 (iab-2) domain, which controls abdA. These two domains can be defined by the phenotypes of rearrangement breakpoints, and by the expression patterns of enhancer traps. In D. virilis, the homeotic cluster is split between Ubx and abd-A, and so the border can also be located by a sequence comparison between species. When the border region is deleted in melanogaster, the flies show a dominant phenotype called Front-ultraabdominal (Fub); the first abdominal segment is transformed into a copy of the second abdominal segment. Thus, the border blocks the spread of activation from the bxd domain into the iab-2 domain.

abd-A regulation by the iab-8 noncoding RNA.

The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This "iab-8 RNA" produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only "in cis." Transcriptional interference with the abd-A promoter is the most likely mechanism.

Functional evolution of cis-regulatory modules at a homeotic gene in Drosophila.

It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox) genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs). How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab) mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules.

Replacement of a Drosophila Polycomb response element core, and in situ analysis of its DNA motifs.

Long-term repression of homeotic genes in the fruit fly is accomplished by proteins of the Polycomb Group, acting at Polycomb response elements (PREs). Here we use gene conversion to mutate specific DNA motifs within a PRE to test their relevance, and we exchange PREs to test their specificity. Previously we showed that removal of a 185 bp core sequence from the bithoraxoid PRE of the bithorax complex results in posteriorly directed segmental transformations. Mutating multiple binding sites for either the PHO or the GAF proteins separately in the core bithoraxoid PRE resulted in only rare and subtle transformations in adult flies. However, when both sets of sites were mutated, the transformations were similar in strength and penetrance to those caused by the deletion of the 185 bp core region. In contrast, mutating the singly occurring binding site of another DNA-binding protein, DSP1 (reportedly essential for PRE-activity), had no similar effect in combination with mutated PHO or GAF sites. Two minimal PREs from other segment-specific regulatory domains of the bithorax complex could substitute for the bithoraxoid PRE core. Our in situ analysis suggests that core PREs are interchangeable, and the cooperation between PHO and GAF binding sites is indispensable for silencing.

MicroRNAs in the Drosophila bithorax complex.

The iab-4 noncoding RNA from the Drosophila bithorax complex is the substrate for a microRNA (miRNA). Gene conversion was used to delete the hairpin precursor of this miRNA; flies homozygous for this deletion are sterile. Surprisingly, this mutation complements with rearrangement breakpoint mutations that disrupt the iab-4 RNA but fails to complement with breaks mapping in the iab-5 through iab-7 regulatory regions. These breaks disrupt the iab-8 RNA, transcribed from the opposite strand. This iab-8 RNA also encodes a miRNA, detected on Northern blots, derived from the hairpin complementary to the iab-4 precursor hairpin. Ultrabithorax is a target of both miRNAs, although its repression is subtle in both cases.

In situ dissection of a Polycomb response element in Drosophila melanogaster.

Genes of the Polycomb group maintain long-term, segment-specific repression of the homeotic genes in Drosophila. DNA targets of Polycomb group proteins, called Polycomb response elements (PREs), have been defined by several assays, but they have not been dissected in their original chromosomal context. An enhanced method of gene conversion was developed to generate a series of small, targeted deletions encompassing the best-studied PRE, upstream of the Ultrabithorax (Ubx) transcription unit in the bithorax complex. Deletions that removed an essential 185-bp core of the PRE caused anterior misexpression of Ubx and posterior segmental transformations, including the conversion of the third thoracic segment toward a duplicate first abdominal segment. These phenotypes were variable, suggesting some cooperation between this PRE and others in the bithorax complex. Larger deletions up to 3 kb were also created, which removed DNA sites reportedly needed for Ubx activation, including putative trithorax response elements. These deletions resulted in neither loss of Ubx expression nor loss-of-function phenotypes. Thus, the 3-kb region including the PRE is required for repression, but not for activation, of Ubx.

Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex.

The three homeotic genes of the bithorax complex (BX-C), Ubx, abd-A and Abd-B control the identity of the posterior thorax and all abdominal segments. Large segment-specific cis-regulatory regions control the expression of Ubx, abd-A or Abd-B in each of the segments. These segment-specific cis-regulatory regions span the whole 300 kb of the BX-C and are arranged on the chromosome in the same order as the segments they specify. Experiments with lacZ reporter constructs revealed the existence of several types of regulatory elements in each of the cis-regulatory regions. These include initiation elements, maintenance elements, cell type- or tissue-specific enhancers, chromatin insulators and the promoter targeting sequence. In this paper, we extend the analysis of regulatory elements within the BX-C by describing a series of internal deficiencies that affect the Abd-B regulatory region. Many of the elements uncovered by these deficiencies are further verified in transgenic reporter assays. Our results highlight four key features of the iab-5, iab-6 and iab-7 cis-regulatory region of Abd-B. First, the whole Abd-B region is modular by nature and can be divided into discrete functional domains. Second, each domain seems to control specifically the level of Abd-B expression in only one parasegment. Third, each domain is itself modular and made up of a similar set of definable regulatory elements. And finally, the activity of each domain is absolutely dependent on the presence of an initiator element.

Cross-regulation among the polycomb group genes in Drosophila melanogaster.

Genes of the Polycomb group in Drosophila melanogaster function as long-term transcriptional repressors. A few members of the group encode proteins found in two evolutionarily conserved chromatin complexes, Polycomb repressive complex 1 (PRC1) and the ESC-E(Z) complex. The majority of the group, lacking clear biochemical functions, might be indirect regulators. The transcript levels of seven Polycomb group genes were assayed in embryos mutant for various other genes in the family. Three Polycomb group genes were identified as upstream positive regulators of the core components of PRC1. There is also negative feedback regulation of some PRC1 core components by other PRC1 genes. Finally, there is positive regulation of PRC1 components by the ESC-E(Z) complex. These multiple pathways of cross-regulation help to explain the large size of the Polycomb group family of genes, but they complicate the genetic analysis of any single member.

Transcription activates repressed domains in the Drosophila bithorax complex.

A series of mutations have been recovered in the bithorax complex of D. melanogaster that transform the first segment of the abdomen into a copy of the second or third abdominal segment. These dominant Ultraabdominal alleles are all associated with P element insertions which are transcribed in the first abdominal segment. The transcripts proceed past the end of the P element for up to 50 kb, extending through the regulatory regions for the second and third abdominal segments. Blocking transcription from the P element promoter reverts the mutant phenotype. Previously identified Ultraabdominal alleles, not associated with P elements, also show abnormal transcription of the same region.