Potential regulatory relationship between the nested gene DDC8 and its host gene tissue inhibitor of metalloproteinase-2

Abstract

Nested genes are fairly common within the mammalian nervous system, yet few studies have examined whether the guest and host genes might be coordinately regulated. Tissue inhibitors of metalloproteinase (TIMPs) inhibit extracellular matrix proteolysis mediated by metzincin proteases. TIMP-2 is the only TIMP not nested within a synapsin gene. It does, however, serve as a host for differential display clone 8 (DDC8), a testis-specific gene whose expression is upregulated during spermatogenesis. Here, we demonstrate that DDC8 is not testis specific. Furthermore, DDC8 expression in nonneural and neural tissues mimics that of TIMP-2, including its upregulation in response to traumatic brain injury, suggesting a potential regulatory relationship. The most striking observation is that the TIMP-2 knockout mouse brain contains TIMP-2 mRNA encoding exons 2-5, which are downstream of DDC8, but not exon 1, which contains the signal sequence and cysteine residue required for MMP inhibition, indicating a functional knockout. That TIMP-2 transcripts in wild-type brain contain DDC8 sequence suggests alternative splicing between the two genes.

Fig. 1. TIMP-2^-/- mice possess TIMP-2 mRNA

A) Genomic structure demonstrating mouse TIMP-2 intron-exon junctions (9). Amino acid resides are indicated within the boxes and the corresponding nucleotide sequence are indicated below (GenBank Accession mRNA: X62622, GeneID: 21858). The shaded area represents the genomic sequence deleted in TIMP-2^-/- mice (57). B) Western blot analysis of adult mouse brain (20 μg crude homogenate) from wild-type (WT) and littermate TIMP-2^-/- (KO) mice, and adult rat brain using antibodies to three distinct TIMP-2 epitopes. In addition to identifying TIMP-2 (28 kDa protein), additional proteins of 78 kDa (Chemicon and Biogenesis) and 62 kDa (Triple Point) are identified (upper blot). TIMP-2 expression is reduced, but not abolished in TIMP-2^-/- mice. Pre-incubation of antibodies with recombinant TIMP-2 protein abrogates (28 kDa TIMP-2 protein) or significantly diminishes (78 and 62 kDa proteins) immunoreactivity (lower blot), thus confirming antibody specificity. C) Reverse zymography of conditioned media from E14 fibroblasts demonstrates that wild-type (+/+), but not knockout (-/-) cells possess MMP-inhibitory activity, indicating a functional knockout. Human recombinant TIMP-2 (hT2) was used as a positive control. D) Northern blot of adult brain (25 μg total RNA) from wild-type, heterozygous (Het) and TIMP-2^-/- littermates hybridized with a full-length TIMP-2 probe. The 1.0 kb transcript is not readily detectable (lower arrowhead), while the 3.5 kb transcript (upper arrowhead) is reduced, but not absent in TIMP-2^-/- mice. An additional ~7.0 kb transcript of unknown origin is also detected in wild-type brain, but is almost absent in TIMP-2^-/- brain. The positions of 28S and 18S rRNA are indicated on the right. PCR of genomic DNA from these animals demonstrating their genotype is shown below the blot. E) PCR with primers specific to each TIMP-2 exon using adult brain from wild-type and 2 TIMP-2^-/- mice (one littermate and one from another litter). No exon 1 product is obtained; however, products for the remaining 4 exons are obtained from TIMP-2^-/- mice. The expression of these exons is reduced approximately 50% relative to wild-type mice. Northern blot analysis of adult brain (25 μg total RNA) hybridized with exon specific probes detects the 3.5 kb transcript in TIMP-2^-/- mice. The different expression level of the 5 exons in wild-type brain suggests TIMP-2 variants with alternative exon usage (see Fig 2A). Cyclophilin was used to verify equal loading of RNA per lane (lower portion blot). PCR of genomic DNA from these animals demonstrating their genotype is shown below the blot.

Fig. 2. TIMP-2 mRNA contains DDC8 sequence

A) EST database analysis indicating potential alternative splicing between DDC8 and its host gene TIMP-2. B) First strand cDNA was primed either with oligo (dT) or with an oligonucleotide specific to TIMP-2 exon 2 (T2). The cDNAs were then amplified with primers within DDC8 exon 3. The oligo (dT) cDNA demonstrates that DDC8 is expressed in adult TIMP-2^-/- (KO) and wild-type (WT) brain. Amplification of the TIMP-2 primed cDNA reveals the presence of TIMP-2 transcripts containing DDC8 sequence in adult TIMP-2^-/- and wild-type mice, and rat brain. TIMP-2 primed cDNA in the absence of reverse transcriptase (no RT) fails to amplify a DDC8 exon 3 product, suggesting the products did not arise from contaminating genomic DNA. C) PCR with oligo (dT) primed adult cDNA amplifies the three DDC8 exons in testis, but only exon 3 in brain indicating that DDC8 mRNA expressed in brain differs from that expressed in testis.

Fig. 3. DDC8 expression is not testis-specific

A) Northern blot analysis of adult wild-type tissues (25 μg total RNA) detects a DDC8 mRNA transcript of 2.0 kb, as well as a diffuse band of ~4.4 kb, only in testis. RNA integrity and equal loading of RNA per lane is demonstrated using the ubiquitously expressed gene cyclophilin (below blot). The positions of 28S and 18S rRNA are indicated on the right. RNA molecular weight standards are indicated on the left. B) Semi-quantitative PCR of oligo (dT) primed cDNA (from 2 μg total RNA) demonstrates that DDC8 is not testis-specific. DDC8 is also abundantly expressed in brain, lung, and kidney. Like DDC8, TIMP-2 expression is abundant in testis, lung, and brain. It should be noted that DDC8 requires 35 cycles while TIMP-2 only 30 cycles to be within the linear amplification range. Cyclophilin (28 cycles of amplification) was used to verify equivalent reverse transcription between samples. C) Semi-quantitative PCR of oligo (dT) primed cDNA (from 1.25 μg total RNA) reveals that DDC8 expression is similar to that of TIMP-2 within the brain, with greatest expression in the brainstem, thalamus, and cerebral cortex. DDC8 expression is greater than TIMP-2 in the cerebellum and hippocampus. Cyclophilin was used to verify equivalent reverse transcription between samples.

Fig. 4. DDC8 expression in the brain is similar to that of TIMP-2

In situ hybridization at the indicated embryonic (A) and postnatal (B) time points of mouse development reveals the spatial distribution of DDC8 expression. DDC8 is expressed throughout brain development, with maximal expression at P21. DDC8 (B_j) is more abundantly expressed than TIMP-2 (D_d) in the adult testis. DDC8 expression (C) mimics that of TIMP-2 (D) in the rat. Sense controls for DDC8 (B_i, B_k) and TIMP-2 (D_c, D_e) demonstrate hybridization specificity in the adult brain and testis, respectively. Hybridized slides were apposed to autoradiographic film for either 3 days (TIMP-2) or 11 days (DDC8). Scale bars = 1 mm.

Fig. 5. DDC8 expression is up-regulated in response to intracranial injury

A) High magnification emulsion-coated sections show that DDC8 expression is similar to TIMP-2 in the mouse retina, but differs in the hippocampus (hippo), cerebral cortex, and testis. Cresyl violet stained section (i) reveals testis morphology. Hybridized slides were exposed to autoradiographic emulsion for either 6 days (TIMP-2) or 22 days (DDC8). B) Coronal sections show that DDC8 expression is up-regulated (arrows) after a penetrating stab injury to the rat brain. Scale bars = 250 μm (A), 1 mm (B).

Fig. 6. DDC8 orthologs exist in multiple species

A) BLASTp, and TBLASTn searches of vertebrate genome and EST databases identified two families of DDC8 genes. One clade, termed DDC8, is supported at 100/100/100 (each value represents the statistical confidence from each of three methods used – see Experimental Design). Three of these genes (e.g., human, mouse, and rat) are nested within the TIMP-2 gene. In addition, a previously unreported clade (here termed DDC8-like), which is supported at a confidence of 70/81/100, is present in more species than the DDC8 clade. Some species have two GI numbers indicated because the sequence similarity extended through more than more gene. B) The most conserved region of the protein sequence alignment for the DDC8 and DDC8-like proteins is shown. This region of mouse DDC8 shows 86% similarity to rat, 60% similarity to macaque, and 57% similarity to human DDC8, but does not correspond to any known functional domain.