Emergence of the PI3-kinase pathway as a central modulator of normal and aberrant B cell differentiation.

Phosphoinositide 3-kinase (PI3K) defines a family of lipid kinases that direct a wide range of cellular processes and cell fate decisions. Since its discovery, and that of its enzymatic antagonist PTEN, much of the focus on PI3K has been on its oncogenic potential. In recent years, studies on PI3K signaling in B lymphocytes have established the importance of this pathway in effecting B cell differentiation and associated molecular events such as V(D)J recombination and class switch recombination. Intriguing new findings also indicate that there is specificity in the PI3K pathway in B cells, including preferential expression or usage of particular PI3K isoforms and counter-regulation by the PTEN and SHIP phosphatases. The role of PI3K adaptor proteins (CD19, BCAP, and TC21) has also undergone revision to reflect both shared and unique properties. The emergence of Foxo1 as a critical PI3K regulatory target for B cell differentiation has united membrane proximal regulatory events orchestrated by PI3K/PTEN/SHIP with key transcriptional targets. Insights into the regulation and impact of PI3K signaling have been brought to bear in new treatments for B cell malignancies, and will also be an important topic of consideration for B cell-dependent autoimmune diseases.

Leukocyte-specific expression of the pp52 (LSP1) promoter is controlled by the cis-acting pp52 silencer and anti-silencer elements.

pp52 (LSP1) is a leukocyte-specific phosphoprotein that binds the cytoskeleton and has been implicated in affecting cytoskeletal remodeling in a variety of leukocyte functions, including cell motility and chemotaxis. The expression of pp52 is restricted to leukocytes by a 549 bp tissue-specific promoter. Here, we show that promoter fragments smaller than the 549 bp pp52 promoter have activity in fibroblasts where pp52 is not normally expressed. Specifically, a truncated construct (+1 to -99) functioned as a basal promoter active in leukocytes and fibroblasts. We identified two upstream regions within the 549 bp pp52 promoter responsible for restricting pp52 promoter activity in fibroblasts. These two regions contained a silencer (pp52 NRE) and an anti-silencer (pp52 anti-NRE) with opposing activities controlling pp52 gene expression. The pp52 NRE was active in both leukocytes and fibroblasts while the pp52 anti-NRE was only active in leukocytes, thereby allowing pp52 gene transcription in leukocytes but not in fibroblasts. The pp52 NRE was localized to an 89 bp DNA segment between -324 and -235 in the 549 bp pp52 promoter and functioned as an active silencer element in a position and orientation independent manner. The pp52 anti-NRE was localized to a 33 bp segment between -383 and -350 of the 549 bp pp52 promoter and acted as an anti-silencer element against the pp52 NRE, but lacked any intrinsic enhancing activity on its own. These findings indicate that the tissue specificity of the pp52 promoter is determined by the pp52 anti-NRE anti-silencer which over-rides the general inhibitory activity of the pp52 NRE silencer.

Cd19-dependent activation of Akt kinase in B-lymphocytes.

CD19 is rapidly phosphorylated upon B-cell antigen receptor (BCR) cross-linking, leading to the recruitment of downstream signaling intermediates. A prominent feature of CD19 signaling is the binding and activation of phosphoinositide 3-kinase (P13K), which accounts for the majority of PI3K activity induced by BCR ligation. Recent findings have implicated activation of the serine/threonine kinase Akt as imparting survival signals in a PI3K-dependent fashion. Using CD19-deficient B-lymphoma cells and mouse splenic B-cells, we show that CD19 is necessary for efficient activation of Akt following cross-linking of surface immunoglobulin or Igbeta. In the absence of CD19, Akt kinase activity is reduced and transient. In addition, coligation of CD19 with surface immunoglobulin leads to augmented Akt activity in a dose-dependent manner. Thus, CD19 is a key regulator of Akt activity in B-cells; as such it may contribute to pre-BCR or BCR-mediated cell survival in vivo.

Silencer elements controlling the B29 (Igbeta) promoter are neither promoter- nor cell-type-specific.

The murine B29 (Igbeta) promoter is B cell specific and contains essential SP1, ETS, OCT, and Ikaros motifs. Flanking 5' DNA sequences inhibit B29 promoter activity, suggesting this region contains silencer elements. Two adjacent 5' DNA segments repress transcription by the murine B29 promoter in a position- and orientation-independent manner, analogous to known silencers. Both these 5' segments also inhibit transcription by several heterologous promoters in B cells, including mb-1, c-fos, and human B29. These 5' segments also inhibit transcription by the c-fos promoter in T cells suggesting they are not B cell-specific elements. DNase I footprint analyses show an approximately 70-bp protected region overlapping the boundary between the two negative regulatory DNA segments and corresponding to binding sites for at least two different DNA-binding proteins. Within this footprint, two unrelated 30-bp cis-acting DNA motifs (designated TOAD and FROG) function as position- and orientation-independent silencers when located directly 5' of the murine B29 promoter. These two silencer motifs act cooperatively to restrict the transcriptional activity of the B29 promoter. Neither of these motifs resembles any known silencers. Mutagenesis of the TOAD and FROG motifs in their respective 5' DNA segments eliminates the silencing activity of these upstream regions, indicating these two motifs as the principal B29 silencer elements within these regions.

Differential interaction of nuclear factors with the leukocyte-specific pp52 promoter in B and T cells.

The leukocyte-specific, cytoskeleton-binding pp52 (LSP-1, WP-34) protein is widely expressed in multiple leukocyte lineages, including B and T lymphocytes, granulocytes, and macrophages. We previously detected a tissue-specific promoter preceding the exon encoding the N terminus of the pp52 leukocyte protein. Here we describe the functional characterization of this promoter and identification of the factors in B and T cells that regulate its activity. The pp52 promoter contains an initiator specifying the unique 5' terminus of pp52 mRNA, tandem pairs of Ets and SP1 motifs, and a lone C/EBP motif. All these motifs are essential and collectively control transcriptional activity. DNA binding studies and Ab supershift assays revealed that different combinations of factors interact with these motifs in B cells vs T cells. The Ets motifs are preferentially bound by PU-1 in B cell extracts from all stages of development, whereas a different Ets family member reacts with these motifs in T cell extracts. The C/EBP motif is bound by Ig/EBP-1 in pre-B cell and T cell extracts, but is replaced by nuclear factor-IL-6beta or a nuclear factor-IL-6beta-Ig/EBP-1 heterodimer in plasmacytoma cell extracts. Despite its reported role as a negative regulator of transcription, Ig/EBP-1 appears to exert a stimulatory effect on this promoter. These findings reveal the features controlling the pp52 promoter in B and T cells and provide the foundation for determining the regulation of this promoter in other leukocyte lineages.

IFN-gamma induces the kappa intron enhancer via an IFN-stimulated response element.

IFN-gamma is a potent inducer of Ig kappa light chain gene transcription in 70Z/3 pre-B cells, but the mechanism of this induction has not been elucidated. The kappa intron enhancer contains a sequence closely resembling the IFN-stimulated response element (ISRE). We have determined that the kappa intron enhancer is IFN-gamma inducible in 70Z/3 cells and that the ISRE is required for this induction. The kappa intron ISRE specifically bound IFN response factor-1 (IRF-1) and the constitutively expressed IRF-2. This ISRE is a multifunctional motif that also binds the LPS-inducible factor kappaBF-A and is located within the kappaBS region, which confers B cell specific activity to this enhancer. However, since the expression of IRF-1 is not restricted to B cells, it must not be sufficient for the induction of kappa transcription. Furthermore, in the pre-B cell line 38B9, which is representative of an earlier stage in pre-B cell development than the 70Z/3 cell line, the kappa intron enhancer was not induced by IFN-gamma despite the activation of IRF-1. These findings suggest that IFN-gamma activation of kappa gene transcription during B cell maturation may be developmentally controlled by elements that restrict the activity of the ISRE within the context of the intron enhancer.

Alternatively spliced exons encode the tissue-specific 5' termini of leukocyte pp52 and stromal cell S37 mRNA isoforms.

The pp52 gene encodes an intracellular, F-actin-binding phosphoprotein (also designated LSP1 and WP34) postulated to function in cytoskeleton dynamics and cell motility. We previously reported that different mRNA isoforms are expressed from this gene in cells of the leukocyte lineage versus mesodermally derived cells. These tissue-specific mRNA isoforms are identical except for 5'-untranslated regions and sequences coding for unique N-termini of 23 and 21 amino acids, respectively. As this is a single-copy gene, we predicted that these tissue-specific mRNA isoforms would be generated by alternative RNA splicing. We report that the unique 5' sequences in these mRNA isoforms are encoded in two separate exons containing ATG initiation codons. These features confirm that the pp52 and S37 mRNA isoforms are generated by alternative RNA splicing and establish that they are independently translated. Other results presented here indicate that the differential expression of these exons in leukocytes versus mesodermally derived cells is regulated at the level of transcription by tissue-specific promoters.

The promoter and 5' flanking sequences controlling human B29 gene expression.

The product of the B-cell-specific B29 gene (B29, Ig beta, CD79b) is essential for Ig-mediated B-cell activation via the B-cell antigen receptor complex (BCR) on human and murine B lymphocytes. To better understand the regulation of this pivotal gene, we have analyzed the human genomic DNA sequence upstream of the B29 ATG start codon for transcriptional control activity. The human B29 gene lacks either a TATA or a CAAT box and transcription is initiated at multiple sites. The minimal promoter of the human B29 gene is contained within a 193-bp region 5' of these multiple start sites. This minimal promoter exhibits B-cell-specific activity and contains SP1, ETS, OCT, and IKAROS/LYF-1 transcription factor motifs. All these motifs are strikingly conserved in sequence and placement relative to the previously characterized murine B29 promoter. Additional upstream gene segments dramatically affected B29 minimal promoter activity. A newly identified motif called the B29 conserved sequence (BCS), found upstream of both human and murine B29 promoters, appears to stimulate B29 transcription through a novel mechanism. A single BCS had little effect either on the minimal B29 promoter or on a heterologous promoter. Instead, the BCS stimulated transcription by counteracting 5' negative regulatory DNA sequences that block the activity of the B29 minimal promoter in its absence. These findings indicate that B29 gene expression is controlled by the complex interplay of positive and negative regulatory elements.

Multiple motifs regulate the B-cell-specific promoter of the B29 gene.

The B-cell-specific B29 and mb1 genes code for covalently linked proteins (B29 or Ig beta and mb1 or Ig alpha, respectively) associated with membrane immunoglobulins in the antigen receptor complex on B cells. We have functionally analyzed the upstream region of the B29 gene and have identified a 164-bp region which comprises the minimal promoter responsible for B-cell-specific transcription. Linker scanning mutagenesis of this minimal promoter has established that both the previously identified octamer motif and a DNA motif that binds an unknown protein factor are critical for B29 gene expression in a pre-B-cell and B-cell line. Further mutations showed that binding motifs for Ets, microB/LyF1, and Sp1 also significantly contributed to the overall activity of the minimal B29 promoter. However, the relative contribution of certain motifs to promoter activity was different in a pre-B versus a B-cell line. The microB/LyF1 motif was necessary for full promoter activity in the pre-B cells but was not required in the B cells.

Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes.

CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation with expression persisting until terminal differentiation into plasma cells. CD19 is a member of the Ig gene superfamily with two extracellular Ig-like domains separated by a non-Ig-like domain, and with an extensive approximately 240 amino acid cytoplasmic domain. In this study, Southern blot analysis revealed that the human and mouse CD19 genes were compact single copy genes. Both the human and mouse CD19 genes were isolated and the nucleotide sequences flanking each exon were determined. Both genes were composed of 15 exons and spanned approximately 8 kilobases (kb) of DNA in human and approximately 6 kb in mouse. The positions of exon-intron boundaries were identical between human and mouse and correlated with the putative functional domains of the CD19 protein. The 200 bp region 5' of the putative translation initiation AUG codon was well conserved in sequence between human and mouse and contained potential transcription regulatory elements. In addition, the 3' untranslated regions (UT) of the CD19 genes following the termination codon were conserved in sequence. The high level of conservation of nucleotide sequences between species in all exons and 5' and 3' UT suggests that expression of the CD19 gene may be regulated in a similar fashion in human and mouse.