Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Novel REST Truncation Mutations Causing Hereditary Gingival Fibromatosis.

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.

Polymorphisms of cytotoxic T lymphocyte-associated antigen-4 and cytokine genes in Taiwanese patients with ankylosing spondylitis.

Cytokines, costimulatory and counter-regulatory molecules play important roles in the regulation of inflammatory response, and are good candidates involved in the development of ankylosing spondylitis (AS). This study investigated the genotypic distribution of proinflammatory cytokines and T-cell negative regulator cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in healthy subjects and AS patients. Genomic DNA was extracted from 143 AS patients and 166 ethnic-matched healthy subjects. Nine polymorphisms within the genes of interleukin-4 (IL-4) (-34T>C, -81A>G, -285C>T and -589T>C), interleukin-6 (IL-6) (-174G>C), interleukin-10 (IL-10) (-592A>C and -819T>C) and CTLA-4 (-318C>T and +49A>G) were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Significantly less AS patients carried the CTLA-4 high-expressing -318 T allele (P = 0.040). The CTLA-4 +49A>G genotypes were associated with circulatory levels of the inflammatory marker C-reactive protein (CRP) (P = 0.022). Our study documented the most complete genetic information of Taiwanese AS patients. The observations that CTLA-4 +49A>G genotypes are associated with circulatory CRP levels and significantly less AS subjects carrying CTLA-4 higher-secretor -318 T allele suggest the level and regulation of inflammation in AS subjects may be pre-determined by and associated with CTLA-4 genotypes.

Prevalence of antibiotics resistance and OXA carbapenemases genes in multidrug-resistant Acinetobacter baumannii isolates in central Taiwan.

This study analyzed the prevalence of antibiotics resistance and the distribution of genes responsible for carbapenems resistance in Acinetobacter baumannii isolates. Clinical A. baumannii isolates were cultured, identified, and collected during the period from May 2007 to February 2009. Antibiotics resistance rates of the clinical isolates were analyzed by antimicrobial susceptibility testing. The distribution of carbapenemase alleles were investigated in the multidrug-resistant (MDR) A. baumannii isolates by multiplex polymerase chain reaction (PCR) techniques. A total of 1,265 independent A. baumannii isolates were identified. Approximately 70% of the clinical isolates were resistant to ampicillin/sulbactam, followed by imipenem, meropenem, cefepime, piperacillin/tazobactam, ceftazidime, and cefoperazone. Overall, 15.18% (192/1,265) of the isolates were characterized as MDR strains. All of the MDR A. baumannii isolates carried the bla (OXA51-like) allele. The detection rate of the bla (OXA23-like) and bla (OXA24-like) alleles was 96.35% (185/192) and 0.52% (1/192), respectively. Most of the isolates (185/192, 96.35%) carried genes which encode more than one carbapenemase. This report demonstrated that approximately 15% of A. baumannii clinical isolates in central Taiwan are MDR strains, with most of them harboring multiple carbapenemases. This study provides updated data regarding the prevalence of beta-lactam resistance and genotyping information of carbapenems resistance of A. baumannii in central Taiwan.

Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation.

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.

Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inactive X chromosomes.

We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.

High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors.

DNA methylation within GC-rich promoters of constitutively expressed X-linked genes is correlated with transcriptional silencing on the inactive X chromosome in female mammals. For most X-linked genes, X chromosome inactivation results in transcriptionally active and inactive alleles occupying each female nucleus. To examine mechanisms responsible for maintaining this unique system of differential gene expression, we have analyzed the methylation of individual cytosine residues in the 5' CpG island of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Methylation analysis of 142 CpG dinucleotides by genomic sequencing was carried out on purified DNA using the cytosine-specific Maxam and Gilbert DNA sequencing reaction in conjunction with ligation-mediated PCR. These studies demonstrate the 5' CpG islands of active and 5-azacytidine-reactivated alleles are essentially unmethylated while the inactive allele is hypermethylated. The inactive allele is completely methylated at nearly all CpG dinucleotides except in a 68-bp region containing four adjacent GC boxes where most CpG dinucleotides are either unmethylated or partially methylated. Curiously, these GC boxes exhibit in vivo footprints only on the active X chromosome, not on the inactive X. The methylation pattern of the inactive HPRT gene is strikingly different from that reported for the inactive X-linked human phosphoglycerate kinase gene which exhibits methylation at all CpG sites in the 5' CpG island. These results suggest that the position of methylated CpG dinucleotides, the density of methylated CpGs, the length of methylated regions, and/or chromatin structure associated with methylated DNA may have a role in repressing the activity of housekeeping promoters on the inactive X chromosome. The pattern of DNA methylation on the inactive human HPRT gene may also provide insight into the process of inactivating the gene early in female embryogenesis.

Nucleosomes are translationally positioned on the active allele and rotationally positioned on the inactive allele of the HPRT promoter.

Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes.

To investigate potential mechanisms regulating the hypoxanthine phosphoribosyltransferase (HPRT) gene by X-chromosome inactivation, we performed in vivo footprinting and high-resolution DNA methylation analysis on the 5' region of the active and inactive mouse HPRT alleles and compared these results with those from the human HPRT gene. We found multiple footprinted sites on the active mouse HPRT allele and no footprints on the inactive allele. Comparison of the footprint patterns of the mouse and human HPRT genes demonstrated that the in vivo binding of regulatory proteins between these species is generally conserved but not identical. Detailed nucleotide sequence comparison of footprinted regions in the mouse and human genes revealed a novel 9-bp sequence associated with transcription factor binding near the transcription sites of both genes, suggesting the identification of a new conserved initiator element. Ligation-mediated PCR genomic sequencing showed that all CpG dinucleotides examined on the active allele are unmethylated, while the majority of CpGs on the inactive allele are methylated and interspersed with a few hypomethylated sites. This pattern of methylation on the inactive mouse allele is notably different from the unusual methylation pattern of the inactive human gene, which exhibited strong hypomethylation specifically at GC boxes. These studies, in conjunction with other genomic sequencing studies of X-linked genes, demonstrate that (i) the active alleles are essentially unmethylated, (ii) the inactive alleles are hypermethylated, and (iii) the high-resolution methylation patterns of the hypermethylated inactive alleles are not strictly conserved. There is no obvious correlation between the pattern of methylated sites on the inactive alleles and the pattern of binding sites for transcription factors on the active alleles. These results are discussed in relationship to potential mechanisms of transcriptional regulation by X-chromosome inactivation.

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.

The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutively expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) within the 5'-flanking region of the human HPRT (hHPRT) gene, which is involved not only in conferring neuronal specificity but also in repressing gene expression in nonneuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation. We also mapped the binding sites for both complexes to a 60-bp region (Ff; positions -510 to -451) which, when analyzed in transfection assays, functioned as a repressor element analogous to the full-length hHPRT-NE sequence. Methylation interference footprintings revealed a minimal unique DNA motif, 5'-GGAAGCC-3', as the binding site for nuclear proteins from both neuronal and nonneuronal sources. However, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the associations of these two complexes. Moreover, UV cross-linking experiments showed that both complexes are formed by the association of several different proteins. Taken together, these data suggest that differential interaction of DNA-binding factors with this regulatory element plays a crucial role in the brain-preferential expression of the gene, and they should lead to the isolation of transcriptional regulators important in neuronal expression of the HPRT gene.

Nucleotide sequence of a region duplicated in Escherichia coli toc mutants.

We have sequenced about 5 kb of the Escherichia coli chromosome downstream from the tolC gene, looking for a topoisomerase gene. This region does not contain a topoisomerase gene.

Cloning and 5'-flanking sequence of a rat cholesterol 7 alpha-hydroxylase gene.

A cholesterol 7 alpha-hydroxylase gene containing 8 kb of the 5'-flanking region and 5 kb of the transcription region which covers exons 1 to 5 was isolated from a rat genomic library. The 2015 bp nucleotide sequence 5'-upstream from the start codon was determined. This promotor region contains many liver-enriched or -specific elements (TGT3, HNF/LF-B1), putative hormone responsive elements (TRE, GRE, RRE or RARE) and ubiquitous transcription factor binding sites (NF-1, OCT-1). In addition, 21 CA repeats which have potential to form the Z-DNA structure were found in this region. These putative regulatory elements and repetitive motifs may play roles in the regulation of cholesterol 7 alpha-hydroxylase gene in the liver. The sequence identity of the rat gene to the human gene in this region is low. Only liver-enriched elements are conserved in the cholesterol 7 alpha-hydroxylase genes.

Analysis of expressed sequence tags from Plasmodium falciparum.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Newly described form of X-linked arthrogryposis maps to the long arm of the human X chromosome.

Arthrogryposis is a heterogeneous birth defect characterized by limitation of movement at multiple joints. One in 3,000 infants is born with arthrogryposis, and at least a third of these cases have a genetic cause. Four distinct types of X-linked arthrogryposis have been reported, and a severe lethal form recently was mapped to Xpll.3-qll.2. We now report an extended family affected with a novel variant of X-linked arthrogryposis that involves only the lower limbs. Linkage analysis with polymorphic DNA markers maps the disease locus in this unique family to the long arm of the human X chromosome between DXS1220 and DXS1205 in Xq23-27.

Evidence that silencing of the HPRT promoter by DNA methylation is mediated by critical CpG sites.

The strong correlation between promoter hypermethylation and gene silencing suggests that promoter methylation represses transcription. To identify methylation sites that may be critical for maintaining repression of the human HPRT gene, we treated human/hamster hybrid cells containing an inactive human X chromosome with the DNA demethylating agent 5-azadeoxycytidine (5aCdr), and we then examined the high resolution methylation pattern of the HPRT promoter in single cell-derived lines. Reactivation of HPRT correlated with complete promoter demethylation. In contrast, the 61 5aCdr-treated clones that failed to reactivate HPRT exhibited sporadic promoter demethylation. However, three specific CpG sites remained methylated in all unreactivated clones, suggesting these sites may be critical for maintaining transcriptional silencing of the HPRT gene. Re-treatment of partially demethylated (and unreactivated) clones with a second round of 5aCdr did not increase the frequency of HPRT reactivation. This is consistent with mechanisms of methylation-mediated repression requiring methylation at specific critical sites and argues against models invoking overall levels or a threshold of promoter methylation. Treatment of cells with the histone deacetylase inhibitor, trichostatin A, failed to reactivate HPRT on the inactive X chromosome, even when the promoter was partially demethylated by 5aCdr treatment, suggesting that transcriptional repression by DNA methylation is unlikely to depend upon a trichostatin A-sensitive histone deacetylase.

Inhibition of the cellular secretion of cholesteryl ester transfer protein by a variant protein formed by alternative splicing of mRNA.

Alternative splicing of mRNA is often used as a regulatory switch, determining whether a functional protein is made or not. The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from high density lipoproteins to other lipoproteins. In addition to the mRNA encoding plasma CETP, human tissues contain an alternatively spliced variant in which exon 9-derived sequences are omitted. To determine a possible regulatory role of alternative splicing, COS cells were co-transfected with full-length and exon 9-deleted cDNAs. The exon 9-deleted protein was poorly secreted and inhibited the secretion of full-length CETP, due to formation of an intracellular heteromeric complex between full-length and exon 9-deleted proteins. The findings suggest a novel use of alternative splicing to generate a poorly secreted protein variant, which complexes with the active form and prevents its secretion by cells.

DNA binding factors for the CpG-rich island containing the promoter of the human X-linked PGK gene.

The gene coding for the glycolytic enzyme phosphoglycerate kinase (PGK-1) is X-linked in mammals and has a G+C-rich 5' region characteristic of several constitutive genes. Despite the fact that PGK-1 is constitutively expressed, it is transcriptionally regulated in female cells by X chromosome inactivation. To study the expression and regulation of the PGK-1 gene, we have analyzed the binding of trans-acting factors to the 5' region of the PGK-1 gene. We detect at least three distinct binding activities that interact in a sequence-specific manner in vitro with at least six different sites in the 5' region. Two of these binding activities generate DNase I-protected footprints centered approximately 360 bp and 130 bp upstream of the transcription start point. We have examined the promoter specificity of the three binding activities in gel mobility-shift assays by competition with cloned promoter fragments of other genes. None of the binding activities interacts exclusively with X-linked promoters. However, one activity binds preferentially to G+C-rich promoters, and another activity appears to bind preferentially to only two of the promoters tested. Previous studies have demonstrated that one HpaII/MspI site, which is included within a footprinted region observed in this study, is fully methylated in the inactive X chromosome and totally unmethylated on the active X chromosome. Competition studies using synthetic oligonucleotides containing 5-methylcytosine at all CpG sites in this region demonstrate that DNA methylation does not significantly alter the affinity between the corresponding binding activity and this binding site.

Autoantibodies from an SLE patient immunostain the Barr body.

To identify specific autoimmune disorders that produce autoantibodies against the mammalian Barr body, sera from 185 autoimmune patients were screened using indirect immunofluorescence on human fibroblasts. Serum from a patient with systemic lupus erythematosus immunostained epi- topes concentrated at the Barr body in female fibroblasts. Such autoantibodies provide a novel tool for characterization of Barr body composition and structure.

Alternative splicing of the human cholesteryl ester transfer protein gene in transgenic mice. Exon exclusion modulates gene expression in response to dietary or developmental change.

The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high density lipoprotein to other lipoproteins. The human DETP gene produces two forms of mRNA, with or without exon 9 (E9)-derived sequences. To study the function and regulation of alternative splicing the CETP gene, transgenic mice were prepared 1) with the metallothionein (mT) promoter driving an E9-deleted construct (mT.CETP(-E9) transgene), and 2) with the natural flanking regions (NFR) controlling expression of genomic sequences which permit alternative splicing of E9 (NFR.CETP(+/-E9) transgene). With zinc induction, the mT.CETP(-E9) transgene gave rise to abundant E9-deleted CETP mRNA in liver and small intestine, but only relatively small amounts of E9-deleted protein were found in plasma. The E9-deleted form of CETP was inactive in lipid transfer and produced no changes in plasma lipoprotein profile. The NFR.CETP(+/-E9) transgene gave rise to full-length (FL) and E9-deleted forms of CETP mRNA in liver and spleen. In response to hypercholesterolemia induced by diet and breeding into an apoE gene knock-out background, the FL CETP mRNA was induced more than the E9-deleted mRNA, resulting in a 2-fold increase in ratio of FL/E9-deleted mRNA. The expression of CETP mRNA was found to be developmentally regulated. In NFR.CETP(+/-E9) transgenic mice CETP mRNA levels were markedly increased in the liver and small intestine in the perinatal period and decreased in adult mice, whereas CETP mRNA in the spleen was low in perinatal mice and increased in adults. The developmental increase in CETP mRNA in the liver and spleen was preceded by an increased ratio of FL/E9-deleted forms. Thus, the E9-deleted mRNA appears to be poorly translated and/or secreted, and the cognate protein is inactive in lipid transfer and lipoprotein metabolism. CETP gene expression was found to be highly regulated in a tissue-specific fashion during development. Increased CETP gene expression during development or in response to hypercholesterolemia is associated with preferential accumulation of the full-length CETP mRNA.