Ulla Hansen

The Human Factors YY1 and LSF Repress the Human Immunodeficiency Virus Type 1 Long Terminal Repeat via Recruitment of Histone Deacetylase 1

ABSTRACT Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

Lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal cells

The E. coli lac operator and repressor were adapted for function in mammalian cells. Plasmids containing an SV40 early region (pSVlacO) or a chloramphenicol acetyl transferase gene (pSVlacOCAT) linked to a hybrid SV40 early promoter bearing a lac operator were tested for function. Identical plasmids lacking an operator (pX-8 and pX-8CAT) were controls. In vitro, early transcription from pSVlacO, but not from pX-8, was inhibited by lac repressor, and repression was overcome by IPTG. Repression of large T synthesis or CAT activity occurred in vivo only when the respective operator-containing plasmid was cotransfected with a plasmid encoding lac repressor, or when the recipient cells stably synthesized lac repressor. IPTG substantially relieved repression in both cases. CAT enzyme repression was paralleled by a decrease in CAT mRNA abundance. Thus regulatory elements of the lac operon function physiologically in mammalian cells.

Alleviation of Histone H1-Mediated Transcriptional Repression and Chromatin Compaction by the Acidic Activation Region in Chromosomal Protein HMG-14

Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes.

Inhibition of the mammalian transcription factor LSF induces S-phase-dependent apoptosis by downregulating thymidylate synthase expression

The thymidylate synthase (TS) gene, which is induced at the G1–S transition in growth‐stimulated cells, encodes an enzyme that is essential for DNA replication and cell survival. Here we demonstrate that LSF (LBP‐1c, CP2) binds to sites within the TS promoter and intronic regions that are required for this induction. Mutation of the LSF binding sites inhibits G1–S induction of mRNA derived from a TS minigene. Furthermore, expression of dominant‐negative LSF (LSFdn) prevents the increase in TS enzyme levels during G1–S, and induces apoptosis in growth‐ stimulated mouse and human cell lines. Such apoptosis can be prevented either by circumventing the TS requirement through addition of low concentrations of thymidine, or by coexpression of the TS gene driven by a heterologous promoter. Induction of apoptosis by LSFdn parallels the process known as thymineless death, which is induced by the TS inhibitor and chemotherapeutic drug 5‐fluorodeoxyuridine. Thus, LSF is a novel regulatory factor that supports progression through S‐phase by targeting a single gene that is critical for cell survival.

Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Kruppel protein.

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.

LSF and NTF-1 share a conserved DNA recognition motif yet require different oligomerization states to form a stable protein-DNA complex.

The mammalian transcription factor LSF (also known as CP2 and LBP-1c) binds as a homo-oligomer to directly repeated elements in viral and cellular promoters. LSF and theDrosophila transcription factor NTF-1 (also known as Elf-1 and Grainyhead) share a similar DNA binding region, which is unlike any established DNA binding motifs. However, we demonstrate that dimeric NTF-1 can bind an LSF half-site, whereas LSF cannot. To characterize further the DNA binding and oligomerization characteristics of LSF, truncation mutants were used to demonstrate that between 234 and 320 amino acids of LSF are required for high affinity DNA binding. Mixing of a truncation mutant with full-length LSF in a DNA binding assay established that the form of LSF that binds DNA is larger than a dimer. Unexpectedly, one C-terminal deletion derivative, partially defective in oligomerization properties, could occupy odd numbers of adjacent, tandem LSF half-sites, unlike full-length LSF. The numbers of DNA-protein complexes formed on multiple half-sites with this mutant indicated that LSF binds DNA as a tetramer, although cross-linking experiments confirmed a previous report concluding that LSF is primarily dimeric in solution. The DNA binding and oligomerization properties of LSF support models depicting novel mechanisms to prevent continual, adjacent binding by a protein that recognizes directly repeated DNA sequences.

ONE EXON OF THE HUMAN LSF GENE INCLUDES CONSERVED REGIONS INVOLVED IN NOVEL DNA-BINDING AND DIMERIZATION MOTIFS

The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors alpha CP2 and LBP-1c and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-1d, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form beta-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.

Nucleosome Positioning and Transcription-associated Chromatin Alterations on the Human Estrogen-responsive pS2 Promoter

The positioning of nucleosomes on a promoter is a significant determinant in its responsiveness to inducing signals. We have mapped the chromatin structure of the human, estrogen-responsive pS2 promoter at nucleotide level resolution within the context of its normal genomic location in human mammary epithelial cells. In vivo digestion by nucleases followed by ligation-mediated polymerase chain reaction analysis revealed two rotationally phased and translationally positioned nucleosomes within the promoter between nucleotide positions −450 and +7. The estrogen response elements at −400 and TATAA box at −35 are each located at the edge of a nucleosome. The two precisely positioned nucleosomes exist in both transformed and nontransformed human mammary epithelial cells, regardless of estrogen receptor status or transcriptional activity of the gene. However, two structural alterations correlate with the transcriptional potential of the promoter. In MCF-7 cells, in which the pS2 promoter is inducible, the chromatin exhibits an increased sensitivity to DNase I in a region of DNA adjacent to the TATAA box and an additional micrococcal nuclease-hypersensitive site in the linker DNA between the two positioned nucleosomes. We were also able to demonstrate that nucleotides −1100 to +10 of the pS2 promoter are sufficient to determine the positioning of these two nucleosomes. Our results establish the structural features of the chromatin covering the pS2 promoter as well as transcriptionally associated alterations, suggesting how the nucleosomal template influences transcriptional regulation by estrogen receptor.

Sequences controlling in vitro transcription of SV40 promoters.

A series of deletion mutants of SV40 were tested for early and late promoter activity in vitro in a transcription extract prepared from HeLa cells. These mutants had previously been characterized for expression in vivo. Transcription in vitro from both the SV40 early and late promoters was strongly dependent on an upstream region of DNA that contains six direct GC repeats. Sequences spanning two or more of these repeats stimulated transcription in a bidirectional fashion, at distances of 50‐200 bp. These sequences may function by mediating the activity of a specific transcriptional factor. Little effect on transcription in vitro was observed upon deletion of the 72‐bp enhancer elements. With this exception, the sequence dependence of early and late transcription in vitro was similar to that observed previously in vivo, both of the region including the GC repeats and of the early TATA sequence.

The ubiquitously expressed DNA-binding protein late SV40 factor binds Ig switch regions and represses class switching to IgA

Ig heavy chain class switch recombination (CSR) determines the expression of Ig isotypes. The molecular mechanism of CSR and the factors regulating this process have remained elusive. Recombination occurs primarily within switch (S) regions, located upstream of each heavy chain gene (except Cδ). These repetitive sequences contain consensus DNA-binding sites for the DNA-binding protein late SV40 factor (LSF) (CP2/leader-binding protein-1c). In this study, we demonstrate by EMSA that purified rLSF, as well as LSF within B cell extracts, directly binds both Sμ and Sα sequences. To determine whether LSF is involved in regulating CSR, two different LSF dominant negative variants were stably expressed in the mouse B cell line I.29 μ, which can be induced to switch from IgM to IgA. Overexpression of these dominant negative LSF proteins results in decreased levels of endogenous LSF DNA-binding activity and an increase in cells undergoing CSR. Thus, LSF represses class switching to IgA. In agreement, LSF DNA-binding activity was found to decrease in whole cell extracts from splenic B cells induced to undergo class switching. To elucidate the mechanism of CSR regulation by LSF, the interactions of LSF with proteins involved in chromatin modification were tested in vitro. LSF interacts with both histone deacetylases and the corepressor Sin3A. We propose that LSF represses CSR by histone deacetylation of chromatin within S regions, thereby limiting accessibility to the switch recombination machinery.