Search

Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP), a broadly applicable biochemical procedure. RNA polymerase II (Pol II) CAST-ChIP identifies ∼1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophilabrain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.

Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila melanogasterembryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, we have undertaken a detailed structure-function analysis of the Knirps protein. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions were identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBPmutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skippedstripe 3 enhancer, is not derepressed in a CtBPmutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities.

Early developmental patterning of the Drosophila embryo is driven by the activities of a diverse set of maternally and zygotically derived transcription factors, including repressors encoded by gap genes such as Krüppel, knirps, giant and the mesoderm-specific snail. The mechanism of repression by gap transcription factors is not well understood at a molecular level. Initial characterization of these transcription factors suggests that they act as short-range repressors, interfering with the activity of enhancer or promoter elements 50 to 100 bp away. To better understand the molecular mechanism of short-range repression, we have investigated the properties of the Giant gap protein. We tested the ability of endogenous Giant to repress when bound close to the transcriptional initiation site and found that Giant effectively represses a heterologous promoter when binding sites are located at −55 bp with respect to the start of transcription. Consistent with its role as a short-range repressor, as the binding sites are moved to more distal locations, repression is diminished. Rather than exhibiting a sharp ‘step-function’ drop-off in activity, however, repression is progressively restricted to areas of highest Giant concentration. Less than a two-fold difference in Giant protein concentration is sufficient to determine a change in transcriptional status of a target gene. This effect demonstrates that Giant protein gradients can be differentially interpreted by target promoters, depending on the exact location of the Giant binding sites within the gene. Thus, in addition to binding site affinity and number, cis element positioning within a promoter can affect the response of a gene to a repressor gradient. We also demonstrate that a chimeric Gal4-Giant protein lacking the basic/zipper domain can specifically repress reporter genes, suggesting that the Giant effector domain is an autonomous repression domain.

DnaA protein of Escherichia coliacts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC.By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitroreplication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriCin which all DnaA boxes are occupied. The last site bound is DnaA box R3. This event may be critical in promoting initiation from oriCas it correlates with in vivoobservations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M. R., Grimwade, J. E., and Leonard, A. C. (1995) EMBO J.14, 5833-5841).

DnaA protein of Escherichia coliis a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriCplasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.