Jamila Horabin - Academia.edu (original) (raw)

Papers by Jamila Horabin

Genetics, Dec 1, 1995

Unlike sex determination in the soma, which is an autonomous process, sex determination in the ge... more Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transfmmer 2 gene but, surprisingly, does not appear to require the sexspecific genes, trunsfonner and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-bthul gene. S EX determination in somatic cells of Drosophila melanogaster is dependent upon an autonomous system that functions transiently in the early embryo (SANCHEZ and NOTHIGER 1983; CLINE 1984). This system measures the relative number of X chromosomes to autosomes (the X/A ratio) in each nucleus and sets the sexual pathway by controlling the transcriptional activity of a special embryonic promoter, Sxl-Pe, of the mas ter regulatory gene, Sex-lethal (Sxl) (-YES et al. 1992). Sxl-Pe is turned on by the signaling system in female (2X/2A) embryos, while it remains off in male (1X/ 2A) embryos. The proteins from the Sxl-Pe mRNAs set in motion an autoregulatory feedback loop that serves to maintain the female-determined state during the remainder of development. In this feedback loop Sxl proteins promote their own expression by directing the productive female-specific splicing of transcripts expressed from the late or maintenance Sxl promoter, Sxl-Pm. In males, the maintenance mechanism also operates at the level of RNA splicing. In the absence of the embryonic proteins, transcripts from Sxl-Pm are spliced to include a male-specific exon that contains in frame translation stop signals that prematurely truncate the open reading frame. The continued splicing of Sxl-Pm transcripts in the nonproductive default pattern during the remainder of the life cycle ensures that the maledetermined state is remembered. Sxl directs subsequent somatic sexual development

Biochimica et biophysica acta, Sep 1, 2014

Genome analysis in several eukaryotes shows a surprising number of transcripts which do not encod... more Genome analysis in several eukaryotes shows a surprising number of transcripts which do not encode conventional messenger RNAs. Once considered noise, these non-coding RNAs (ncRNAs) appear capable of controlling gene expression by various means. We find Drosophila sex determination, specifically the master-switch gene Sex-lethal (Sxl), is regulated by long ncRNAs (>200 nt). The lncRNAs influence the dose sensitive establishment promoter of Sxl, Sxl Pe , which must be activated to specify female sex. They are primarily from two regions, R1 and R2, upstream of Sxl Pe and show a dynamic developmental profile. Of the four lncRNA strands only one, R2 antisense, has its peak coincident with Sxl Pe transcription, suggesting it may promote activation. Indeed, its expression is regulated by the X chromosome counting genes, whose dose determines whether Sxl Pe is transcribed. Transgenic lines which ectopically express each of the lncRNAs show they can act in trans, impacting the process of sex determination but also altering the levels of the other lncRNAs. Generally, expression of R1 is negative whereas R2 is positive to females. This ectopic expression also results in a change in the local chromatin marks, affecting the timing and strength of Sxl Pe transcription. The chromatin marks are those deposited by the Polycomb and Trithorax groups of chromatin modifying proteins, which we find bind to the lncRNAs. We suggest the increasing numbers of non-coding transcripts being identified are a harbinger of interacting networks similar to the one we describe.

Genetics, Mar 1, 2018

P REVIOUSLY, we suggested that before female sexual identity in Drosophila is fully established, ... more P REVIOUSLY, we suggested that before female sexual identity in Drosophila is fully established, the default state of expression is male and X chromosome genes are expressed at a higher, twofold male level (Gladstein et al. 2010). This enhanced expression, driven by the male-specific lethal (msls) genes, which target the X chromosome for hypertranscription [reviewed in Lucchesi and Kuroda (2015)], was proposed to consolidate female sex determination, given that the primary sex determination genes and their target, the Sex-lethal (Sxl) master-switch, all reside on the X. However, a recent publication has claimed that the Drosophila sex determination process does not utilize the MSL complex, as it found no female-lethal genetic interactions between the msls and Sxl, or the primary sex determination genes (Erickson 2016). Erickson did not address the molecular data, which included demonstrating reduced transcription at both the initiation (Sxl Pe) and maintenance (Sxl Pm) promoters of Sxl in several MSL complex mutant backgrounds. The genetics tested for synergism in female lethality from combining reduced msl dose with mutations in sex determination genes:

Fly, 2012

Requirement of male-specific dosage compensation in Drosophila females-implications of early X ch... more Requirement of male-specific dosage compensation in Drosophila females-implications of early X chromosome gene expression.

PubMed, 1996

mRNAs from the early Sex-lethal promoter, Sxl-Pe, encode embryonic Sxl proteins that function to ... more mRNAs from the early Sex-lethal promoter, Sxl-Pe, encode embryonic Sxl proteins that function to activate the Sxl autoregulatory loop. They do so by directing the female-specific splicing of the first transcripts expressed from the late or maintenance promoter, Sxl-Pm. The early promoter is located, however, upstream not downstream of the translation terminating male-specific exon, L3, and upstream of the second Sxl-Pm exon, L2. If the Sxl proteins expressed from Sxl-Pe are to provide the mechanism for bypassing the normal requirement for Sxl protein in the female-specific splicing of transcripts from Sxl-Pm, then what is the mechanism for skipping L2 and L3 in the processing of transcripts from Sxl-Pe? In the studies reported here, we have generated a report construct to examine the splicing of Sxl-Pe transcripts. Our results indicate that neither specific maternal products, Sxl protein, nor an X chromosome to autosome ratio of 1 are required for the processing of the embryonic mRNAs. We also found that none of the three genes, snf, virilizer, and fl(2)d, which when mutated alter the female-specific processing of Sxl-Pm transcripts, alter the generation of the early splice. Skipping to intervening exons to generate an open reading frame that will encode the Sxl early proteins appears to be an intrinsic property of initiating the early Sxl RNAs within the first intron of the Sxl-Pm maintenance transcription unit.

Development, Jul 1, 1993

In addition to controlling somatic sexual development in Drosophila melanogaster, the Sex-lethal ... more In addition to controlling somatic sexual development in Drosophila melanogaster, the Sex-lethal (Sxl) gene is required for proper differentiation of female germ cells. To investigate its role in germ-line development, we have examined the expression of Sxl in wild-type ovaries and ovaries that are defective in early steps of germ cell differentiation. As in the soma, the basic mechanism for on/off regulation of Sxl relies on sex-specific processing of its transcripts in germ cells. One class of female-sterile mutations, which includes fs(1)1621 and the tumorousovary-producing allele of the ovarian tumor gene, otu 1 , is defective in the splicing process. These mutants have germ lines with high amounts of Sxl RNA spliced in the male mode and a severe reduction of protein levels in the germ cells. Another class of female-sterile mutations produces a phenotype similar to that seen in fs(1)1621 and otu 1 but appears to express normal levels of Sxl protein in the germ cells. However, this second class does not show the changes in protein distribution normally observed in wild-type germ cells. In the wild-type germarium, the non-differentiated germ cells show a strong cytoplasmic accumulation of Sxl protein followed, as the germ cells differentiate, by a dramatic reduction and redistribution of the protein into nuclear foci. Interestingly, two female-sterile alleles of Sxl, Sxl f4 and Sxl f5 belong to the second class, which shows persistent cytoplasmic accumulation of Sxl protein. These Sxl femalesterile mutants encode an altered protein indicating that Sxl regulates processes that eventually lead to the changes in Sxl protein distribution. Lastly, we demonstrate that during the final stages of oogenesis several mechanisms must operate to prevent the progeny from inheriting Sxl protein. Conceivably, this regulation safeguards the inadvertent activation of the Sxl autoregulatory feedback loop in the male zygote.

Journal of Molecular Biology, Apr 1, 1986

We have cloned the gene I sequence of the filamentous bacteriophage f1 downstream from the lambda... more We have cloned the gene I sequence of the filamentous bacteriophage f1 downstream from the lambda leftward promoter on a plasmid that also contains the temperature-sensitive lambda repressor, cI857. Temperature induction of gene I protein (pI) resulted in rapid cessation of growth. This inhibition appears to involve a rapid decrease in synthesis of host protein and RNA. The ability of pI to cause this inhibition is not dependent on thioredoxin, a host factor that is necessary for phage morphogenesis and has been shown by genetic data to interact with pI. The inhibition does not appear to be mediated by the amino half of the protein, as induction of an identical plasmid construction of an amber mutant positioned two-thirds along gene I, does not affect cell growth. Analysis of the transcription products from the cloned gene I confirmed previous suggestions that a transcription terminator exists in the amino-terminal portion of the gene. In addition, there is no detectable promoter activity in the 152 bases immediately upstream from the gene. These data and the inability to overproduce pI argue for down-regulation of pI production. Radioactive labeling of proteins in maxi-cells and normal Escherichia coli cells identifies pI as a protein of about 39,000 Mr that partitions with the cell envelope. Pulse-chase experiments suggest that pI is not processed to any appreciable extent.

Cell, Apr 1, 1991

Sex-lethal is a binary switch gene that controls all aspects of Drosophila sexual dimorphism. It ... more Sex-lethal is a binary switch gene that controls all aspects of Drosophila sexual dimorphism. It must be active in females and inactive in males. The on/off regulation reflects alternative RNA splicing in which full-length proteins are produced only in females. Here we investigate the role of Sxl in maintaining sexual pathway commitments. By ectopic expression of a female Sxl cDNA in transgenic male flies, we show that Sxl protein induces a rapid switch from male- to female-specific splicing. The ectopically expressed Sxl protein wil trans-activate an endogenous wild-type Sxl gene. This establishes a feedback loop in which Sxl proteins induce their own synthesis by directing the female-specific splicing of Sxl transcripts. We conclude that the female determined state is maintained by Sxl through positive autoregulation, while the male determined state is maintained by default.

Chromosome Research, Oct 23, 2013

The study of long noncoding RNAs (lncRNAs) is still in its infancy with more putative RNAs identi... more The study of long noncoding RNAs (lncRNAs) is still in its infancy with more putative RNAs identified than those with ascribed functions. Defined as transcripts that are longer than 200 nucleotides without a coding sequence, their numbers are on the rise and may well challenge protein coding transcripts in number and diversity. lncRNAs are often expressed at low levels and their sequences are frequently poorly conserved, making it unclear if they are transcriptional noise or bonafide effectors. Despite these limitations, inroads into their functions are being made and it is clear they make a contribution in regulating all aspects of biology. The early verdict on their activity, however, suggests the majority function as chromatin modifiers. A good proportion show a connection to disease highlighting their importance and the need to determine their function. The focus of this review is on lncRNAs which influence developmental processes which in itself covers a large range of known activities.

Epigenetics & Chromatin, Apr 30, 2015

Background: The small non-histone protein Heterochromatin protein 1a (HP1a) plays a vital role in... more Background: The small non-histone protein Heterochromatin protein 1a (HP1a) plays a vital role in packaging chromatin, most notably in forming constitutive heterochromatin at the centromeres and telomeres. A second major chromatin regulating system is that of the Polycomb/trithorax groups of genes which, respectively, maintain the repressed/activated state of euchromatin. Recent analyses suggest they affect the expression of a multitude of genes, beyond the homeotics whose alteration in expression lead to their initial discovery. Results: Our data suggest that early in Drosophila development, HP1a collaborates with the Polycomb/trithorax groups of proteins to regulate gene expression and that the two chromatin systems do not act separately as convention describes. HP1a affects the levels of both the Polycomb complexes and RNA polymerase II at promoters, as assayed by chromatin immunoprecipitation analysis. Deposition of both the repressive (H3K27me3) and activating (H3K4me3) marks promoted by the Polycomb/trithorax group genes at gene promoters is affected. Additionally, depending on which parent contributes the null mutation of the HP1a gene, the levels of the H3K27me3 and H3K9me3 silencing marks at both promoters and heterochromatin are different. Changes in levels of the H3K27me3 and H3K9me3 repressive marks show a mostly reciprocal nature. The time around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, appears to be a transition/decision point for setting the levels. Conclusions: We find that HP1a, which is normally critical for the formation of constitutive heterochromatin, also affects the generation of the epigenetic marks of the Polycomb/trithorax groups of proteins, chromatin modifiers which are key to maintaining gene expression in euchromatin. At gene promoters, deposition of both the repressive H3K27me3 and activating H3K4me3 marks of histone modifications shows a dependence on HP1a. Around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, a pivotal decision for the level of silencing appears to take place. This is also when the embryo organizes its genome into heterochromatin and euchromatin. A balance between the HP1a and Polycomb group silencing systems appears to be set for the chromatin types that each system will primarily regulate.

Journal of Bacteriology, Sep 1, 1983

An Escherichia coli DNA fragment containing the structural gene serU132 for the nonsense suppress... more An Escherichia coli DNA fragment containing the structural gene serU132 for the nonsense suppressor tRNA2sr was identified and purified by being cloned into a plasmid vector. Information obtained from DNA sequence analysis was used to select a serU132 fragment for insertion downstream from the bacteriophage A PL promoter in two pBR322-A derivatives. In nonsense mutant strains bearing the resulting serU132 hybrid plasmids, the presence of the A c1857 repressor gene carried on the same plasmid or in a prophage genome permits thermal regulation of suppressor synthesis. MATERIALS AND METHODS Nomenclature. The supD-allele used in these studies was supD32, from the Garen strain S26rleA-(8). Since this allele was identified as the gene encoding an amber-suppressing seryl tRNA, the wild-type locus is now designated serU+ and the nonsense suppressor allele is designated serU132. The corresponding gene products are tRNAser and tRNAs". In accord with the format used for the E. coli map (2), Sup' and Supdenote the suppressor-negative and suppressor-positive phenotypes, respectively. Materials. Carrier-free 32p, was obtained from New England Nuclear Corp. and incorporated into [-y

Development, Jul 15, 2001

Sex-lethal is the Drosophila melanogaster sex determination master switch. It is also required in... more Sex-lethal is the Drosophila melanogaster sex determination master switch. It is also required in female germ cells to control mitosis and meiotic recombination. As early germ cells mature, distinct changes in both Sex-lethal protein levels and localization occur. By manipulating the levels of Hedgehog and making germline clones of components in the hedgehog signaling pathway, we demonstrate that Hedgehog affects the nuclear translocation of Sex-lethal and the levels of the protein in early germ cells. This effect is mediated primarily through degradation. Consistent with the Hedgehog pathway regulating Sex-lethal, we find Sex-lethal in a complex with Fused and Costal-2, both downstream components of the pathway. This is the first demonstration that downstream components of the Hedgehog signaling pathway regulate a target other than Cubitus interruptus.

Molecular and Cellular Biology, Dec 1, 1993

The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual develo... more The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual development in Drosophila melanogaster, is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the Sxl proteins directs the splicing machinery to produce functional mRNAs. We have used germ line transformation to analyze the mechanism of default and regulated splicing. Our results demonstrate that a blockage mechanism is employed in Sxl autoregulation. However, in contrast to transformer, in which Sxl appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in autoregulation. (i) Multiple cis-acting elements, both upstream and downstream of the male exon, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the Sxl protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role.

Humana Press eBooks, 2007

concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitatio... more concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

Molecular and Cellular Biology, Mar 1, 1993

In Drosophila melanogaster, sex determination in somatic cells is controlled by a cascade of gene... more In Drosophila melanogaster, sex determination in somatic cells is controlled by a cascade of genes whose expression is regulated by alternative splicing [

Genetics, Aug 1, 2000

It has been suggested that sexual identity in the germline depends upon the combination of a nona... more It has been suggested that sexual identity in the germline depends upon the combination of a nonautonomous somatic signaling pathway and an autonomous X chromosome counting system. In the studies reported here, we have examined the role of the sexual differentiation genes transformer (tra) and doublesex (dsx) in regulating the activity of the somatic signaling pathway. We asked whether ectopic somatic expression of the female products of the tra and dsx genes could feminize the germline of XY animals. We find that Tra F is sufficient to feminize XY germ cells, shutting off the expression of male-specific markers and activating the expression of female-specific markers. Feminization of the germline depends upon the constitutively expressed transformer-2 (tra-2) gene, but does not seem to require a functional dsx gene. However, feminization of XY germ cells by Tra F can be blocked by the male form of the Dsx protein (Dsx M). Expression of the female form of dsx, Dsx F , in XY animals also induced germline expression of female markers. Taken together with a previous analysis of the effects of mutations in tra, tra-2, and dsx on the feminization of XX germ cells in XX animals, our findings indicate that the somatic signaling pathway is redundant at the level tra and dsx. Finally, our studies call into question the idea that a cellautonomous X chromosome counting system plays a central role in germline sex determination.

Developmental Biology, 2003

The sex determination master switch, Sex-lethal, has been shown to regulate the mitosis of early ... more The sex determination master switch, Sex-lethal, has been shown to regulate the mitosis of early germ cells in Drosophila melanogaster. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of Ornithine Decarboxylase Antizyme, was isolated. gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. We also show that Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis.

fragmentfromaphagecontainingthe Sxl region from exon 2 to exon 4 was then ligatedupstream of the ... more fragmentfromaphagecontainingthe Sxl region from exon 2 to exon 4 was then ligatedupstream of the exon 4-lacZ sequences to reconstruct theregulated region ofSxl. TheSxlminiconstruct also served asthe substrate for site-directed mutagenesis (29) of the poly-pyrimidine tract at the male exon 3' splice site (see Fig. 5)and for the deletion constructs described below.TheuniqueNcoIsite in the male exonwasused to deletemostofthe male exonsequence (by exonuclease III) for theconstruct diagrammed in Fig. 3. The ends were madebluntwith Klenowfragment digested withPstI, and the fragmentfrom exon 2 to the shortened male exon was cloned up-stream of lacZ sequences to create the single-intron con-struct. The male exon-lacZ junction was determined bysequencing with a primer in lacZ sequences. The constructdiagrammedin Fig. 3 had 11 bpofthe exon from the secondAGsplice site of the male exon.Togenerate the tra-like constructs, two approaches wereused. In the first, the shortened male exon fragme...

Journal of Biological Chemistry, 1988

A 55-amino acid segment, normally present between residues 241 and 295 of the 348-residue gene I ... more A 55-amino acid segment, normally present between residues 241 and 295 of the 348-residue gene I protein of the filamentous bacteriophage f l , acts as an internal signal sequence for gene I protein or, when present in fusion proteins, for EcoRI endonuclease or alkaline phosphatase. The resulting proteins are inserted so that they span the membrane with sequences on the amino side of the 55-residue segment in the cytoplasm and those near the carboxy side outside the cytoplasmic membrane. The presence of these proteins in the membrane results in the rapid inhibition of cell growth, probably from a loss of the membrane potential. We describe some of the elements in this 55-residue segment that appear to be crucial for its interaction with the membrane.