Long human CHGA flanking chromosome 14 sequence required for optimal BAC transgenic "rescue" of disease phenotypes in the mouse Chga knockout

Abstract

Chromogranin A (CHGA) plays a catalytic role in formation of catecholamine storage vesicles and also serves as precursor to the peptide fragment catestatin, a catecholamine secretory inhibitor whose expression is diminished in the hypertensive individuals. We previously reported the hypertensive, hyperadrenergic phenotype of Chga-/- knockout (KO) mice and rescue by the human ortholog. In the present study, we compare two humanized CHGA mouse models. Into the Chga null background, by bacterial artificial chromosome transgenesis human CHGA transgene has been introduced. Both lines have the complete approximately 12 kbp CHGA gene integrated stably in the genome but have substantial differences in CHGA expression, as well as consequent sympathochromaffin biochemistry and physiology. A mouse model with longer-insert HumCHGA31 displays integration encompassing not only CHGA but also long human flanking sequences. This is in contrast to mouse model HumCHGA19 with limited flanking human sequence co-integrated. As a consequence, HumCHGA19 mice have normal though diminished pattern of spatial expression of CHGA, and 14-fold lower circulating CHGA, with failure to rescue KO phenotypes to normalcy. In the longer-insert HumCHGA31 mice, catecholamine secretion, exaggerated responses to environmental stress, and hypertension were all alleviated. Promoter regions of the transgenes in both HumCHGA19 and HumCHGA31 display minimal CpG methylation, weighing against differential "position effects" of integration, and thus suggesting that lack of cis elements required for optimal CHGA expression occurs in HumCHGA19 mice. Such "humanized" CHGA mouse models may be useful in probing the physiological consequences of variation in CHGA expression found in humans, with consequences for susceptibility to hypertension and cardiovascular disease.

Fig. 1.

Structures of the integrated human chromogranin A (CHGA) transgenes. A: schematic depicting the ∼220 kbp BAC (bacterial artificial chromosome) construct used to create “humanized” CHGA mice. The insert comprises the ∼211 kbp chromosome 14 sequence encompassing the entire 12.1 kbp human CHGA gene, as well as ∼44 kbp upstream and ∼155 kbp downstream sequence. B: integrated sequence in the 2 founders Tg(CHGA:RPC11-862G15)19Smv;Chga+/− (“Tg19”) and Tg(CHGA:RPC11-862G15)31Smv; Chga +/− (“Tg31”) is shown. Dotted lines indicate the extent of flanking sequence incorporated in each transgene. The founder Tg31 has almost the entire BAC insert integrated stably into the mouse genome. In founder Tg19 with limited flanking sequences, ∼41 kbp upstream and ∼19 kbp downstream are integrated along with the entire CHGA gene.

Fig. 2.

Single copy human CHGA transgene integration into the mouse genome. A: slot blot analysis of DNA. 1 μg or 100 ng duplicate samples of genomic DNA from human (diploid) or from F1 pups of founders Tg19 and Tg31 were hybridized with a 500 bp human CHGA-specific probe (160,219–159,374 of forward strand of human chromosome 14) with no cross-hybridization to mouse Chga. B: a densitometric scan of slot blot A revealed that the intensity of the bands in case of the F1 pups matches about half that of the same mass of human DNA, indicative of a single copy integration of CHGA per diploid mouse genome. Therefore F1 pups of founders Tg19 and Tg31 have 1 copy of the transgene CHGA and 2 copies of Chga. C: Southern blot analysis of DNA. DNA (10 μg) from Tg19, Tg31, or human DNA was restricted with the 8-base cutter XmnI (which does not cut within CHGA), run on a 1% agarose-1× TBE gel, blotted, and subjected to Southern analysis. Again the same probe as described in A was used. Human diploid DNA (h) has 2 copies of CHGA, giving rise to a single band that hybridizes with the probe. A single band of the same size indicates 1 copy insertion in mice derived from founders Tg19 and Tg31, confirming results of the slot blot analysis. D: CHGA mRNA expression analysis by real-time PCR. cDNAs from adrenal gland (adr), brain [hippocampus (hip), frontal cortex (fron cor), striatum (str), cerebellum (cer), pituitary (pit), hypothalamus (hyp)], pancreas (pan), ovary (ova), spleen (spl), and liver (liv) tissues were probed for human chromogranin A gene expression in both transgenic strains Tg19 and Tg31 bearing a single copy of the human CHGA transgene as well as the endogenous mouse Chga alleles. The CHGA transgene is expressed exclusively in neuroendocrine tissues and not in the liver and spleen. Tg19 has low expression compared with Tg31 whose values lie entirely to the left of the line of identity.

Fig. 3.

Chromogranin A expression in “humanized” CHGA mice: protein and mRNA. A: human CHGA protein expression. Immunoblot analysis (representative blot). Adrenal protein extracts of HumCHGA31, knockout (KO), wild-type (WT), and HumCHGA19 were probed with a rabbit polyclonal antibody raised against the catestatin domain of human CHGA (amino acids 352–372). Probing with an antibody against actin normalized the blot. B: bar diagram quantifies the densitometric scan of the blot. C: human CHGA mRNA expression. Real-time PCR analysis for human CHGA expression in HumCHGA31 (n = 4) and HumCHGA19 (n = 5) adrenal and brain stem tissues indicates significantly higher expression in HumCHGA31 compared with HumCHGA19. D: human CHGA ELISA. Plasma from WT, HumCHGA19, and HumCHGA31 mice (each group n = 8) analyzed by ELISA, using a monoclonal mouse anti-human CHGA antibody. The capturing antibody mouse monoclonal anti-human antibody B4E11 is specific for human CHGA; there was no signal over baseline for WT (+/+) or KO (−/−) mouse plasma. This antibody specificity for human CHGA has been documented (7, 28) Significantly lower levels of circulating human CHGA were observed in HumCHGA19 compared with HumCHGA31 plasma.

Fig. 4.

Microanatomy of chromogranin A expression: confocal micrographs of mouse adrenal glands. As depicted in F, each of the pictures (A–E) has 4 subpanels: Top left, red nuclear staining (TO-PRO 3 iodine, 640λ); top right, green CHGA staining (Alexa 488λ); bottom left, phase contrast showing medulla and cortex regions; bottom right, composite of the previous 3 panels. The bar indicates 20 μm. A: control staining, with WT adrenal slice incubated with 2° antibody in the absence of 1° CHGA antibody. B: WT; C: KO; D: HumCHGA31 transgene; E: HumCHGA19 transgene. Magnification is ×800.

Fig. 5.

Preferential “rescue” of elevated blood pressure to normalcy by the longer human CHGA transgene Tg31. Higher systolic blood pressure (SBP) and diastolic blood pressure (DBP) are observed in KO, HumCHGA19, and heterozygote (mouse Chga +/−, Het) mice, indicating that lower levels of CHGA expression in HumCHGA19 or Het (+/−) fail to completely rescue the KO (−/−) BP to normalcy. HumCHGA31 mice in contrast have BP indistinguishable from WT (Chga+/+). The age of the mice was 10–12 wk. WT, n = 8; KO, n = 12; HumCHGA31, n = 21; HumCHGA19, n = 30; heterozygotes (Het) n = 17. A: SBP. Overall (1-way ANOVA across all groups), the SBP difference was significant at P < 0.0001. Bonferroni-corrected P values are shown for individual pair-wise comparisons. B: DBP. Overall (1-way ANOVA across all groups), the SBP difference was significant at P < 0.0001. Bonferroni-corrected P values are shown for individual pair-wise comparisons.

Fig. 6.

Rescue of catecholamine levels to normalcy by the longer human CHGA transgene Tg31. Plasma catecholamines were measured in 10–12 wk old mice, n = 8/group. A and B: comparison of norepinephrine and epinephrine levels in WT and KO mice. C and D: humanized mice catecholamines compared with WT. HumCHGA19 mice have elevated norepinephrine and epinephrine in the plasma compared with WT and HumCHGA31 mice. HumCHGA31 mice with 2 copies of the human CHGA gene effectively compensate for loss of Chga alleles and rescue the catecholamine levels to normalcy.

Fig. 7.

BP response to immobilization stress. WT vs. knockout SBP normal and poststress. Humanized CHGA transgenic mice: SBP changes in response to immobilization stress. Chronic stress causes the SBP to be elevated significantly in WT, KO, and HumCHGA31 mice. Age of the mice is 4 mo, with n = 6 WT and KO, and n = 8 humanized mice studied under both control and stressed conditions. HumCHGA19 mice have higher SBP at the start of the experiment at age 4 mo, and chronic stress does not further elevate SBP.

Fig. 8.

HumCHGA19 and HumCHGA31 transgenes: expression and location. A: human CHGA. Local genomic region in the BAC insert. The image was created at http://genome.ucsc.edu using the assembly February 2009 human reference sequence (GRCh37). Displayed are local genes, regions of sequence conservation across vertebrate species, and repeat regions. The limits of the BAC clone RP11862G15 contained in the integrated transgene of HumCHGA19 and HumCHGA31 are depicted in gray at the bottom. B: genes contained in the BAC clone RP11862G15. The BAC clone RP11862G15 spans human chromosome 14 region 93,345,420–93,556,350. Therefore, ITPK1 gene is incomplete and 5′ end of the gene (∼26 kb) is deleted in RP11862G15. Four coding genes along with CHGA are co-inserted in line HumCHGA31. AK024887 is a noncoding RNA. C: expression of flanking genes co-inserted with CHGA. Differential expression of CHGA flanking genes is observed in the two strains. In adrenal gland, brain (total), testis, lung, and spleen, human CHGA mRNA was detected by RT-PCR in both HumCHGA19 and HumCHGA31 strains. D: methylation status of the human CHGA promoter in transgene. Both transgenic lines HumCHGA31 and HumCHGA19 were analyzed for the degree of methylation in the first 5 CpG sites within the proximal promoter of the transgene in adrenal DNA by Pyrosequencing analysis. CHGA promoter of both the transgenes display low methylation status, consistent with expression of the genes. As a genome-wide control, mouse LINE-1 sequence methylation was also assayed, and expectedly displayed higher methylation, consistent with “silencing.”