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L L Wallrath

Publications and source records attributed to L L Wallrath.

At least 19 recordsLinked to original sources
PubMed2000

Down-regulation of HP1Hsalpha expression is associated with the metastatic phenotype in breast cancer.

We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsalpha expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells (poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsalpha, but not HP1Hsbeta or HP1Hsgamma, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsalpha staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsalpha localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsalpha expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsalpha protein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsalpha in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsalpha in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsalpha normally silences genes involved in breast cancer invasion and metastasis.

Breast↗
PubMed2000

The fourth chromosome of Drosophila melanogaster: interspersed euchromatic and heterochromatic domains.

The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50-75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

Animals↗
PubMed2000

Drosophila telomeric transgenes provide insights on mechanisms of gene silencing.

A significant fraction of most eukaryotic genomes is packaged into chromatin that is not permissive for gene expression. This silent chromatin is typically located near centromeres and telomeres and has fascinated scientists for more than 70 years, yet many questions remain unanswered. Part of the difficulties in studying silent chromatin at the molecular level is the repetitive nature of the DNA sequences in these regions. To overcome this problem, Drosophila stocks carrying in vitro designed transgenes inserted within silent chromatin have been generated. Molecular analysis of these transgenes has shed light on the nature of the chromatin structure within these regions and provided insights on the mechanisms of gene silencing. This review will focus on recent studies using telomeric transgenes. The results from these studies suggest that nuclear organization plays a role in gene silencing and that silencing is the result of a block early in the process of transcription initiation.

Animals↗
PubMed1999

Heterochromatic silencing of Drosophila heat shock genes acts at the level of promoter potentiation.

In a variety of organisms, genes placed near heterochromatin are transcriptionally silenced. In order to understand the molecular mechanisms responsible for this block in transcription, high resolution in vivo chromatin structure analysis was performed on two heat shock genes, hsp26 and hsp70. These genes normally reside in euchromatin where GAGA factor and RNA Pol II are present on the promoter prior to heat shock induction. P-element transformation experiments led to the identification of stocks in which these two genes were inserted within heterochromatin of the chromosome 4 telomeric region. These transgenes exhibit silencing that is partially suppressed by mutations in the gene encoding HP1. Micrococcal nuclease analysis revealed that the heterochromatic transgenes were packaged in a more regular nucleosome array than when located in euchromatin. High resolution DNase I analysis demonstrated that GAGA factor and TFIID were not associated with these promoters in heterochromatin; potassium permanganate experiments showed a loss of Pol II association. Taken together, these data suggest that occlusion of trans-acting factors from their cis- acting regulatory elements leading to a block in promoter potentiation is a mechanism for heterochromatin gene silencing.

Animals↗
PubMed1999

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

Animals↗
PubMed1998

Characterization of sequences associated with position-effect variegation at pericentric sites in Drosophila heterochromatin.

In a variety of organisms, euchromatic genes brought into juxtaposition with pericentric heterochromatin show position-effect variegation (PEV), a silencing of gene expression in a subset of the cells in which the gene is normally expressed. Previously, a P-element mobilization screen identified transgenic Drosophila stocks showing PEV of an hsp70-white+ reporter gene; transgenes in many of these stocks map to the chromocenter of polytene chromosome. A screen at an elevated temperature identified two stocks that under standard culture temperatures show complete repression of the hsp70-white+ transgene. The transgenes in both cases map to the chromocenter of polytene chromosomes. Different types of middle repetitive elements are adjacent to seven pericentric transgenes; unique sequences are adjacent to two of the perimetric transgenes. All of the transgenes show suppression of PEV in response to a mutation in the gene encoding heterochromatin protein 1 (HP1). This suppression correlates with a more accessible chromatin structure. The results indicate that a pericentric transgene showing PEV can be associated with different types of DNA sequences, while maintaining a common association with the chromosomal protein HP1.

ATP-Binding Cassette Transporters↗
PubMed1998

Unfolding the mysteries of heterochromatin.

The function of heterochromatin has not been well understood. Recent studies, however, demonstrate that heterochromatin is essential for proper chromosome behavior. The silencing of euchromatic genes by heterochromatin has been exploited to understand the molecular nature of heterochromatin. Mutations that either suppress or enhance gene silencing exist within chromatin structural proteins and modifying enzymes. Interactions between some of these proteins have been demonstrated, suggesting a complicated picture of heterogeneous silencing complexes that are counteracted by protein-modifying machinery.

Animals↗
PubMed1996

DNA representation of variegating heterochromatic P-element inserts in diploid and polytene tissues of Drosophila melanogaster.

Position-effect variegation (PEV) is the mosaic expression of a euchromatic gene brought into juxtaposition with heterochromatin. Fourteen different transformed Drosophila melanogaster lines with variegating P-element inserts were used to examine the DNA levels of these transgenes. Insert sites include pericentric, telomeric and fourth chromosome regions. Southern blot analyses showed that the heterochromatic hsp26 transgenes are underrepresented 1.3- to 33-fold in polytene tissue relative to the endogenous euchromatic hsp26 gene. In contrast, the heterochromatic hsp26 transgenes are present in approximately the same copy number as the endogenous euchromatic hsp26 gene in diploid tissue. It appears unlikely that DNA loss could account for the lack of gene expression in diploid tissues seen with these examples of PEV.

ATP-Binding Cassette Transporters↗
PubMed1995

The role of a positioned nucleosome at the Drosophila melanogaster hsp26 promoter.

The regulatory region of Drosophila melanogaster hsp26 includes a positioned nucleosome located between the two DNase I hypersensitive (DH) sites that encompass the critical heat shock elements (HSEs). To test the role of this nucleosome in regulated expression, transgenic flies containing hsp26-lacZ fusion genes with alterations in the nucleosome-associated region have been generated. The positioned nucleosome is associated with a DNA sequence that does not itself contain any critical regulatory elements for heat shock-inducible expression. The nucleosome-associated sequence can be deleted, reversed, duplicated or replaced by a random sequence with no significant effect on DH site formation and gene expression. Analyses of hsp26 and hsp70 transgenes with spacing changes within the promoter region indicate that the location of the (CT)n.(GA)n elements dictates the location of DH site formation. Wrapping the DNA between the regulatory elements around a nucleosome is as effective for gene expression as placing the regulatory elements close to each other. A loss of inducible gene expression was observed when the nucleosome-associated DNA was replaced with sequences which appear to misdirect nucleosome placement. The results indicate considerable flexibility in the spacing between DH regulatory sites.

Animals↗
PubMed1995

Position effect variegation in Drosophila is associated with an altered chromatin structure.

A euchromatic gene placed in the vicinity of heterochromatin by a chromosomal rearrangement generally exhibits position effect variegation (PEV), a clonally inherited pattern showing gene expression in some somatic cells but not in others. The mechanism responsible for this loss of gene expression is investigated here using fly lines carrying a P element containing the Drosophila melanogaster white and hsp26 genes. Following mobilization of the P element, a screen for variegation of white expression recovered inserts at pericentric, telomeric, and fourth chromosome regions. Previously identified suppressors of PEV suppressed white variegation of pericentric and fourth chromosome inserts but not telomeric inserts on the second and third chromosomes. This implies a difference in the mechanism for gene repression at telomeres. Heat shock-induced hsp26 expression was reduced from pericentric and fourth chromosome inserts but not from telomeric inserts. Chromatin structure analysis revealed that the variegating inserts showed a reduction in accessibility to restriction enzyme digestion in the hsp26 regulatory region in isolated nuclei. Micrococcal nuclease digests showed that pericentric inserts were packaged in a more regular nucleosome array than that observed for euchromatic inserts. These data suggest that altered chromatin packaging plays a role in PEV.

Animals↗
PubMed1994

Insensitivity of the present hsp26 chromatin structure to a TATA box mutation in Drosophila.

The role of the TATA element in establishing the chromatin structure and inducible transcription of the Drosophila melanogaster hsp26 gene has been analyzed. An hsp26/lacZ fusion gene with a mutant promoter, in which the TATA box sequence TATAAA was changed to CCCAAA, was introduced into Drosophila by P-element transformation. The mutation had little effect on formation of the preset chromatin structure observed prior to induction. However, the mutation dramatically reduced transcription levels following heat shock. Northern analysis indicated that weak, inducible expression of the mutant promoter occurred within the same period of heat shock as for the normal promoter, suggesting that TFIID was associated with the mutant promoter prior to heat shock. Biochemical analysis showed that the mutant promoter still bound TFIID in vitro, but with 3-5-fold less affinity than the normal promoter. DNase I footprinting revealed that the conformation of the TFIID-DNA complex differed significantly from that of the normal promoter. These results indicate that alterations in the conformation or the stability of the TFIID-DNA complex drastically reduce the level of induction, but do not dramatically affect chromatin structure formation. Formation of the requisite chromatin structure is either independent of, or highly tolerant of, changes in the TFIID-DNA complex.

Animals↗
PubMed1994

Architectural variations of inducible eukaryotic promoters: preset and remodeling chromatin structures.

The DNA in a eukaryotic nucleus is packaged into a nucleosome array, punctuated by variations in the regular pattern. The local chromatin structure of inducible genes appears to fall into two categories: preset and remodeling. Preset genes are those in which the binding sites for trans-acting factors are accessible (i.e. in a non-nucleosomal, DNase I hypersensitive configuration) prior to activation. In response to the activation signal, positive factors bind to cis-acting regulatory elements and trigger transcription with no major alterations in the chromatin structure of the promoter region. In contrast, remodeling genes are those in which some of the required cis-acting regulatory elements are packaged into nucleosomes. The nucleosomes must be perturbed in response to an activation signal in order for the trans-acting factors to gain access to cis-acting elements; a chromatin remodeling process which forms DNase I hypersensitive sites must occur. In both cases, precise positioning of nucleosomes along the promoter region of a gene appears to be critical for appropriate regulation of expression.

Animals↗
PubMed1994

Nucleosome positioning and gene regulation.

Recent genetic and biochemical studies have revealed critical information concerning the role of nucleosomes in eukaryotic gene regulation. Nucleosomes package DNA into a dynamic chromatin structure, and by assuming defined positions in chromatin, influence gene regulation. Nucleosomes can serve as repressors, presumably by blocking access to regulatory elements; consequently, the positions of nucleosomes relative to the location of cis-acting elements are critical. Some genes have a chromatin structure that is "preset," ready for activation, while others require "remodeling" for activation. Nucleosome positioning may be determined by multiple factors, including histone-DNA interactions, boundaries defined by DNA structure or protein binding, and higher-order chromatin structure.

Base Sequence↗
PubMed1993

(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene.

Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

Animals↗