Construction and analysis of cells lacking the HMGA gene family

Abstract

The high mobility group A (HMGA) family of non-histone chromosomal proteins is encoded by two related genes, HMGA1 and HMGA2. HMGA proteins are architectural transcription factors that have been found to regulate the transcription of a large number of genes. They are also some of the most commonly dysregulated genes in human neoplasias, highlighting a role in growth control. HMGA1 and HMGA2 have also been found to stimulate retroviral integration in vitro. In this study, we have cloned chicken HMGA1, and used the chicken DT40 B-cell lymphoma line to generate cells lacking HMGA1, HMGA2 and both in combination. We tested these lines for effects on cellular growth, gene control and retroviral integration. Surprisingly, we found that the HMGA gene family is dispensable for growth in DT40 cells, and that there is no apparent defect in retroviral integration in the absence of HMGA1 or HMGA2. We also analyzed the activity of approximately 4000 chicken genes, but found no significant changes. We conclude that HMGA proteins are not strictly required for growth control or retroviral integration in DT40 cells and may well be redundant with other factors.

Figure 1

(A) Alignment of human and chicken HMGA1b. Gray boxes indicate position of A-T hook DNA binding domains. (B) Partial map of chicken HMGA1 locus and the locus after recombination with a knockout construct. Gray boxes indicate regions of genomic DNA included in the knockout construct. Hatched box represents HMGA1 coding exons (note: intron–exon boundaries were not mapped). Cross-hatched box represents antibiotic selection cassette. P indicates region of probe used on Southern blot to screen for homologous recombinants. (C) Partial map of 5′ end of chicken HMGA2. Representations are as in (B). (B) and (C) are not drawn to scale.

Figure 2

Southern blots showing genotype of various HMGA cell lines. Arrows indicate size of wild-type and knockout alleles. Cell lines are: (1) wild-type DT40, (2) A1-1, (3) A1-2, (4) A1-3, (5) A2-1, (6) A2-2, (7) A1/A2-1 and (8) A1/A2-2. (A) Southern blot of SphI-digested genomic DNA probed with DNA from region indicated in Figure 1B. (B) Southern blot of KpnI-digested genomic DNA probed with DNA from region indicated in Figure 1C.

Figure 3

Expression of HMGA1 and HMGA2 in knockout cell lines. RNA samples are as follows: (1) wild type DT40, (2) A1-1, (3) A1-2, (4) A1-3, (5) A2-1, (6) A2-2, (7) A1/A2-1 and (8) A1/A2-2. (A) Northern showing HMGA1 expression. The ethidium bromide stained 28S rRNA image is included to show equal loading. (B) RT–PCR analysis of HMGA2 expression.

Figure 4

Growth rate of HMGA knockout lines. Wild-type DT40, closed diamonds; A1-1, closed squares; A1-2, closed triangles; A1-3, open circles; A2-1, crossed lines; A2-2, closed circles; A1/A2-1, open squares; A1/A2-2, open triangles.

Figure 5

Single round infection of HMGA knockout lines with HIV-based lentiviral vectors. (A) Diagram of the HIV-based lentiviral vector construct. LTR, long terminal repeat; Ψ, genome packaging signal; cPPT, central polypurine tract; CMVie, CMV immediate early promoter; Luc+, luciferase coding region; WPRE, woodchuck hepatitis virus post transcriptional regulatory element. (B) Strategy for infecting DT40 cell lines. Lentiviral vectors were generated by transfection of 293T cells with vector-producing constructs. Supernatant was collected from cells and filtered to remove cellular debris. DT40 cells were spin-infected and incubated for 2–3 days at 37°C. After the incubation period, cells were lysed and luciferase activity was quantified. (C) Relative luciferase activity after infection with lentiviral vectors. All values are normalized to wild-type DT40 luciferase activity. Integrase (+) values represent the average of three experiments. Integrase (–) values represent the average of two experiments. Cell lines are: (1) wild-type DT40, (2) A1-1, (3) A1-2, (4) A1-3, (5) A2-1, (6) A2-2, (7) A1/A2-1 and (8) A1/A2-2.

Figure 6

Spreading infection of RCASBP(C)GFP. Wild-type DT40, closed diamonds; A1-1, closed squares; A1-2, closed triangles; A1-3, closed circles; A2-1, open diamonds; A2-2, open squares; A1/A2-1, open triangles; A1/A2-2, open circles.