Patrick Matthias

Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA)

The octamer sequence ATGCAAAT is found in the promoters of immunoglobulin (Ig) heavy and light chain genes and in the heavy chain enhancer and is a major determinant of the cell type specific expression of Ig genes in B cells. An apparent paradox is that the same sequence serves as an upstream promoter or enhancer element in a variety of housekeeping genes such as the histone H2B and U snRNA genes. The differential usage of this regulatory sequence motif is thought to be mediated by different species of octamer binding proteins. One species of 100 kd, designated OTF‐1, is present in all cell types and may exert its activating function only when it can interact with additional adjacent transcription factors. The lymphoid cell specific octamer binding protein of 60 kd (OTF‐2A) specifically stimulates Ig promoters which consist essentially of a TATA‐box and an octamer sequence upstream of it. Here we present evidence for yet another B cell specific octamer binding protein of 75 kd (OTF‐2B). From several findings, including the absence of OTF‐2B (but not OTF‐2A) from a lymphocyte line that cannot respond to the IgH enhancer, we propose a role of the novel octamer factor in the long range activation by the IgH enhancer. We have used the proteolytic clipping bandshift assay (PCBA) technique to distinguish the three different forms found in B cells. This analysis indicates that the 75 kd‐species OTF‐2B is closely related to the 60 kd species OTF‐2A.

Cell-type-specificity elements of the immunoglobulin heavy chain gene enhancer

A strong transcriptional enhancer was created by oligomerization of a short segment from the immunoglobulin heavy chain (IgH) enhancer. This segment was analyzed in parallel for biological activity in vivo and factor binding in vitro. In transfection experiments the oligomerized segment stimulates transcription in a cell type‐specific manner similar to the entire IgH enhancer. Transfections of mutants identified two sequence motifs whose integrity is required for efficient and cell type‐specific activity of this enhancer. The first is a sequence suggested previously to be bound by a factor in vivo, and the second is a highly conserved decanucleotide which also occurs in Ig variable gene promoters. The ability of these two sequence motifs to bind proteins in vitro was tested by band shift assays. Under our in vitro conditions we could not detect proteins binding to the in vivo footprint region. However, we found protein factors binding to the decanucleotide. A ubiquitous form of this factor is present in every cell line analyzed. Additional variants are detected exclusively in cells where the IgH enhancer and the segment thereof are active. Elimination of the decanucleotide motif is not only a strong down mutation in vivo but also abolishes binding of all factor variants in vitro. Thus our data suggest that the two enhancer motifs analyzed are involved in positive rather than negative control of transcription.

Octamer transcription factors bind to two different sequence motifs of the immunoglobulin heavy chain promoter.

All promoters of immunoglobulin heavy chain genes contain three conserved sequence motifs: a heptamer motif CTCATGA, an octamer motif ATGCAAAT, and a TATA box. We show that, despite their different sequences, both the heptamer and the octamer motif are bound by the same octamer transcription factors (Oct factors, also referred to as OTFs), namely the lymphoid‐specific proteins Oct‐2A and Oct‐2B, as well as the ubiquitous protein Oct‐1. Even though binding to the octamer motif is stronger, a single heptamer motif can bind Oct proteins and mediate transcriptional activity in lymphoid cells. Furthermore, factor binding to the octamer motif facilitates binding to the nearby heptamer motif. We propose that the heptamer element plays a role early in B‐cell differentiation to ensure that the heavy chain promoters are transcriptionally activated before the light chain promoters, which do not contain the heptamer motif.

Transcription factor Oct-2A contains functionally redundant activating domains and works selectively from a promoter but not from a remote enhancer position in non-lymphoid (HeLa) cells.

In non‐lymphoid cells such as HeLa cells, ectopic expression of the lymphocyte‐specific transcription factor Oct‐2A can activate reporter genes whose promoters consist of a single octamer sequence (ATTTGCAT) upstream of a TATA box. While the factor is strongly active in a promoter position, it tails as an enhancer factor: an enhancer consisting of multiple copies of the octamer sequence placed downstream of the reporter gene is not active in HeLa cells, even at high concentration of Oct‐2A. In B lymphoid cells, however, the same enhancer is highly active. This could mean that an additional factor is required for enhancer activation in B cells. Furthermore, we have tested the transcriptional activation potential of Oct‐2A with a series of N‐terminal and C‐terminal deletions. We show that a glutamine‐rich domain near the N‐terminus is required for full activity. Otherwise, large segments of the N‐terminal half or the entire C‐terminal region are dispensable in our assay, as long as the deletions do not impinge on the conserved POU domain which is sufficient for DNA binding. While N‐terminal and C‐terminal regions can functionally compensate for each other, a combined deletion that only retains the POU domain is a strong down mutation. We also find that activity depends on the promoter structure of the reporter gene: the POU domain by itself shows some activity with a promoter where the octamer sequence is located very close to the TATA box, but no activity with another promoter construction where the octamer sequence is located further upstream. The two promoters also respond differently to the deletion of the glutamine‐rich stretch important for transcriptional activation. From these experiments we consider it likely that the natural octamer factor variants can selectively activate the different naturally occurring octamer‐containing promoters.

Both immunoglobulin promoter and enhancer sequences are targets for suppression in myeloma-fibroblast hybrid cells.

When immunoglobulin (Ig)‐producing B cells are fused with fibroblastic cells, expression of Igs is suppressed by a mechanism that selectively abolishes transcription of Ig genes. The suppression is also maintained in proliferating hybrids. We have used gene transfer followed by cell fusion to study this phenomenon further. Here we report that expression of a rearranged Ig heavy chain gene, stably integrated into a myeloma genome, is completely suppressed upon fusion with fibroblasts by a mechanism that is equally active on the endogenous myeloma lambda light chain gene. To define regulatory sequences within the Ig transcriptional unit that are involved in this down‐regulation, we examined the transcriptional contributions of the IgH chain gene enhancer and the kappa light chain gene promoter individually by linking them to a heterologous reporter gene. Mouse myeloma cells were stably transformed with such test constructs and subsequently fused with mouse fibroblasts. To avoid any significant loss of chromosomes, hybrid cells were isolated shortly after fusion by fluorescence‐activated cell sorting, and proliferating hybrids were harvested within 2‐3 weeks. On the basis of RNase protection mapping of cytoplasmic RNA, and of nuclear run‐on assays we showed that both the kappa light chain promoter and the IgH chain enhancer contain regulatory information that is made redundant or is suppressed in the hybrid environment.

Myeloma χ gene transcription is blocked upon fusion with fibroblasts

Abstract Numerous studies on somatic cell hybrids have shown that expression of tissue-specific functions can be suppressed as a consequence of fusion with cells that do not express the given functions. We have further investigated this phenomenon, using as a model system the regulation of expression of χ light chain genes in intraspecific hybrids between mouse myeloma cells and mouse fibroblasts. Hybrids containing only one genome equivalent from each parent cell (1s:1s) were isolated by fluorescence-activated cell sorting from within 10 h after fusion, and they were grown for no more than 16 days thereafter in order to ensure maximum integrity of the genomic constitution. Here we report that in hybrid cells, χ gene transcription was specifically turned off as demonstrated by nuclear run-on assays performed on 16-day-old proliferating hybrids. Furthermore, a mechanism affecting mRNA stability may also contribute, at least initially, to the rapid depletion of cytoplasmic χ transcripts, observed during the first few hours after fusion. Suppression was dominant and could not be overridden by increasing the relative myeloma ploidy at either the heterokaryon or the synkaryon stage. Nor could suppression be relieved by treating hybrids with Cycloheximide.

A transcriptional terminator between enhancer and promoter does not affect remote transcriptional control.

Enhancers stimulate transcription of RNA polymerase II-transcribed genes in an orientation-independent manner and over long distances. This stimulation is known to be associated with an increased polymerase density over the linked gene. However, many aspects of the exact mechanism of remote gene control remain to be elucidated. Based on some reports on RNA polymerase I transcription, we wanted to test whether RNA polymerase II enters at the enhancer and from there proceeds towards the promoter while synthesizing unstable transcripts (“scanning/readthrough transcription” model). For this, we have inserted a complete terminator region from the mouse β-globinmaj gene between the SV40 enhancer and the rabbit β-globin promoter. In contrast to what the model predicts, insertion of the terminator had no affect on remote enhancer action. Furthermore, we have determined the RNA polymerase density over the spacer DNA between enhancer and promoter, and over the reporter gene, by means of the so-called run-on transcription assay. We find very low transcription of the spacer, but high transcription of the globin reporter gene. Thus, our data are not consistent with a scanning/readthrough transcription mechanism where RNA polymerase II would move from the enhancer to the promoter while transcribing the intervening spacer DNA. These and other findings are compatible with a model where enhancer and promoter are brought into close proximity, perhaps with concomitant looping out of the intervening DNA.

Cloning of Sequence-Specific DNA-Binding Proteins by Screening λ cDNA Expression Libraries with Radiolabelled Binding-Site Probes

The isolation of DNA-binding, transcription factors has been facilitated by a novel strategy that depends on the functional expression in E. coli of high levels of the DNA-binding domain (DBD) of the factor studied. This procedure (Singh et al., 1988, Vinson et al., 1988) , which is essentially a modification of the original antibody screening of λ gtll libraries (Young & Davis 1983) , can be outlined as follows (see Fig. 1) : A cDNA expression library is constructed in an inducible prokaryotic expression vector such as the bacteriophage vectors λ gt11 or λ ZAPII. Both vectors produce proteins fused with the N-terminal portion of s-galactosidase, and in each case synthesis of the fusion protein is under the control of the lac repressor. After plating of the library, the phage plaques are blotted onto filters which had previously been soaked in a solution of IPTG, a lactose analogue. IPTG inactivates the lac repressor, and thus induces in situ expression of the fusion proteins from the λ recombinants. The presence of the desired fusion protein is then detected by incubating the filters with a radiolabelled DNA probe containing the binding site for the desired factor, under conditions comparable to those used for footprint or gel-retardation experiments. After washing and autoradiography of the filters, the phages deemed positive are eluted and replated. Parallel filters are then prepared, induced for protein expression and probed with either the genuine binding-site probe, or, ideally a point-mutated version of it known to abolish binding in vitro (Fig. 1).