Germline Genome Research Articles

Epidemiology, Genetics and Pathogenesis of Pituitary Neuroendocrine Tumors

As the molecular pathology of pituitary cell development and the process of tumorigenesis in this organ continues to advance, it is recommended that pituitary neuroendocrine tumors(PitNETs)be classified based on three lineage-specific transcription factors(PIT1, Tpit, and SF1). In the hyperaging society of Japan, the number of cases traditionally classified as nonfunctioning PitNETs is increasing, and it is possible that some of these tumors may be associated with tumors that are known to exhibit aggressive behavior. The molecular pathological background of PitNET development is highly variable, and its pathogenesis in many cases remains unclear. As genomic analysis of PitNETs progresses, it is becoming increasingly clear that abnormalities in germline and somatic cell genomes contribute to our understanding of their etiology but do not explain most of them. Epigenetic modifications, such as deoxyribonucleic acid methylation and histone modifications(methylation and acetylation), are thought to be intricately related to tumorigenesis.

Parents' expectations, preferences, and recall of germline findings in a childhood cancer precision medicine trial.

Germline genome sequencing in childhood cancer precision medicine trials may reveal pathogenic or likely pathogenic variants in cancer predisposition genes in more than 10% of children. These findings can have implications for diagnosis, treatment, and the child's and family's future cancer risk. Understanding parents' perspectives of germline genome sequencing is critical to successful clinical implementation. A total of 182 parents of 144 children (<18 years of age) with poor-prognosis cancers enrolled in the Precision Medicine for Children with Cancer trial completed a questionnaire at enrollment and after the return of their child's results, including clinically relevant germline findings (received by 13% of parents). Parents' expectations of germline genome sequencing, return of results preferences, and recall of results received were assessed. Forty-five parents (of 43 children) were interviewed in depth. At trial enrollment, most parents (63%) believed it was at least "somewhat likely" that their child would receive a clinically relevant germline finding. Almost all expressed a preference to receive a broad range of germline genomic findings, including variants of uncertain significance (88%). Some (29%) inaccurately recalled receiving a clinically relevant germline finding. Qualitatively, parents expressed confusion and uncertainty after the return of their child's genome sequencing results by their child's clinician. Many parents of children with poor-prognosis childhood cancer enrolled in a precision medicine trial expect their child may have an underlying cancer predisposition syndrome. They wish to receive a wide scope of information from germline genome sequencing but may feel confused by the reporting of trial results.

Genome editing of human embryos for research purposes: Japanese lay and expert attitudes.

Background: Multiple surveys of the general public and experts on human genome editing have been conducted. However, many focused only on editing in clinical applications, with few regarding its use for basic research. Given that genome editing for research purposes is indispensable for the realization of clinical genome editing, understanding lay attitudes toward genome editing in research, particularly using human embryos, which is likely to provoke ethical concerns, is helpful for future societal discussion. Methods: An online survey was conducted with Japanese laypeople and researchers to ascertain their views regarding human genome editing for research purposes. Participants were queried about their acceptance as a function of the target of genome editing (germ cells, surplus IVF embryos, research embryos, somatic cells); then, those who answered "acceptable depending on the purpose" were asked about their acceptance in the context of specific research purposes of genome editing. Participants were also asked about their expectations and concerns regarding human genome editing. Results: Replies were obtained from 4,424 laypeople and 98 researchers. Approximately 28.2-36.9% of the laypeople exhibited strong resistance to genome editing for research purposes regardless of their applications. In contrast, 25.5% of the researchers demonstrated resistance only to genome editing in research embryos; this percentage was substantially higher than those concerning the other three targets (5.1-9.2%). Approximately 50.4-63.4% of laypeople who answered "acceptable depending on the purpose" approved germline genome editing for disease research; however, only 39.3-42.8% approved genome editing in basic research to obtain biological knowledge. In contrast, the researchers displayed a lower degree of acceptance of germline genome editing for research purposes related to chronic diseases (60.9-66.7%) than for other research purposes (73.6-90.8%). Analysis of responses concerning expectations and concerns indicated that laypeople who would not accept genome editing of human embryos did not necessarily worry about "instrumentalization of the embryo." They also had substantially low expectations for recognized advantages of genome editing, including "advances in science" and "reduction of intractable diseases," compared with other groups of respondents. Conclusion: The assumptions shared among experts in conventional bioethical debates and policy discussions on human genome editing are not self-evident to laypeople.

Safety and Ethical Considerations of CRISPR/Cas9-based Human Germline Genome Editing

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 adapted from natural immune defense systems of bacteria and archaea can edit genes in numerous organisms including humans. This has revolutionized genetic engineering and can help better understand genetic diseases as well as potentially correct causative mutations. Current CRISPR/Cas9-based genome editing is focused on either somatic cells or germline cells. In contrast to somatic cell genome editing, germline genome editing raises significant ethical issues as the genomic modifications in those reproductive cells by CRISPR/Cas9 can potentially be passed on to future generations. In this article, we focus on discussion of safety and ethical issues of CRISPR/Cas9-based germline genome editing in humans from several aspects including off-targets, Immunogenicity, autonomy, religion and eugenics.

When Is Something an Alternative? A General Account Applied to Animal-Free Alternatives to Animal Research.

The first "R" from animal research ethics prescribes the replacement of animal experiments with animal-free alternatives. However, the question of when an animal-free method qualifies as an alternative to animal experiments remains unresolved.Drawing lessons from another debate in which the word "alternative" is central, the ethical debate on alternatives to germline genome editing, this paper develops a general account of when something qualifies as an alternative to something. It proposes three ethically significant conditions that technique, method, or approach X must meet to qualify as an alternative to Y: (1) X must address the same problem as Y, under an appropriate description of that problem; (2) X must have a reasonable chance of success, compared to Y, in solving the problem; and (3) X must not be ethically unacceptable as a solution. If X meets all these conditions, its relative advantages and disadvantages determine whether it is preferable, indifferent, or dispreferable as an alternative to Y.This account is then applied to the question of whether animal-free research methods qualify as alternatives to animal research. Doing so breaks down the debate around this question into more focused (ethical and other) issues and illustrates the potential of the account.

Genomic analysis in patients with soft tissue sarcoma to examine gene mutations involved in chromosome segregation, mitotic spindle assembly, and degradation.

e23523 Background: Soft tissue sarcoma (STS) is a rare cancer with few drug discovery. Recent genomic analysis of STS revealed a high degree of chromosomal instability (CIN) including genome-wide copy number alterations, aneuploidy, whole genome doubling and chromothripsis. In STS patients, we have reported extensive somatic LOH in tumors, a hallmark of CIN, represented by haploid of germline mutations/variants in cancer-related genes. CIN of STS implicates abnormalities in chromosome segregation, spindle assembly and degradation in mitosis but few studies have examined the impact of gene mutations involved in the pathological mitotic processes in STS. Methods: We recruited 155 patients with metastatic/recurrent STS (135 female and 20 male, mean age 51 at analysis, 100 LMS, 19 LPS, 4 ESS, 3 UPS, 3 AS, 3 MPT and others) with information of familial cancer burden. Whole exome sequencing and analysis were performed in both blood and tumor samples as described in 2018ASCO. Results: STS Patients with tumors harboring less than 33% somatic LOH (n = 54) in a total of somatic and LOH mutations/variants in 595 COSMIC genes have a better prognosis than those (34-66%, n = 49, 67-100%, n = 52) with more LOH genes (5-year OS; 72% vs 53% or 40%, p = 0.0499, p = 0.0023, respectively). LOH was associated with neither TMB nor MSI status but with a total number of somatic and germline mutations (R = 0.874). Of the genes involved in chromosome segregation and mitosis, we found that a family of ARHGAP genes which play a role in the spindle degradation and Aurora A kinase activation was most frequently mutated in tumor and germline genomes (n = 102 in a total of 155, n = 71 in 100 LMS). Damaging ARHGAP mutations/variants are correlated with higher LOH values (54.1±2.8%, n = 102 vs. 33.1±3.8%, n = 53, mean±SE, p &lt; 0.001, for LMS, 63.3±2.9%, n = 71 vs. 36.7±5.4%, n = 29, mean±SE, p &lt; 0.001) and poor prognosis of patients (5-year OS; 48% n = 102 vs. 71% n = 53, p = 0.0107). We also found damaging mutations/variants in a family of CAPN (calpain) genes involved in cleavage of the cohesion ring for chromosome separation (n = 73 in 155) and CENPT gene essential for bridging between kinetochore and microtubules via NDC80 (n = 43 in 155). For CENPT, loss of wild type allele at codons encoding functional Ser121 and Arg122 was found in 10 patients with LMS and DSRCT. Damaging CAPN mutations/variants are correlated with higher LOH values (60.6±3.0%, n = 73 vs. 34.8±3.2%, n = 82, mean±SE, p &lt; 0.001). Conclusions: This study demonstrates extensive genomic abnormalities involved in the pathways of chromosome segregation and mitosis in STS. Recent studies, including ourselves, confirmed overexpression of KIF18A, NDC80 and NUF2 in STS, lethal targets for KIF18A and CHD (Calponin Homology Domain) inhibitors in CIN tumors. Together, our results will open a new avenue evolving treatment landscape of STS.

Germline sex determination: How males and females tame transposons in their own ways

The piRNA pathway represses transposon activity to protect the germline genome for future generations. Anew study shows how germline sex determination influences the production of different piRNAs in male and female germ cells.

Family communication and results disclosure after germline sequencing: A mixed methods study

ObjectiveResearch on family communication of germline genome sequencing (GS) results (versus of genetic results after targeted genetic testing) is still emerging, yet potentially complex results increase the importance of communicating risk to relatives. Promoting equity by ensuring patients have sufficient health literacy to interpret results is important in this context. This study aimed to identify cancer patients’ perceived importance of result disclosure, predictors of perceptions, and perspectives on family communication. MethodsThis explanatory-sequential, cross-sectional mixed-methods study involved participants (n = 246) completing a questionnaire and (n = 20) a semi-structured interview. Ordinal logistic regressions determined associations between potential predictors and perceived importance of result disclosure. Interview transcripts were analysed thematically using a constant-comparative approach. ResultsMore participants intended disclosing to nuclear (77.4%) than to extended family (42.7%). More than half (59.3%) felt results were family information; 62.7% believed it was important to disclose results to family members. Nuclear and extended family communication scores and education level were significantly positively associated with perceived importance of disclosure (p < 0.05). Six qualitative themes were identified: i) Responsibility to inform, ii) Choice, iii) Autonomy, iv) Family Communication, v) Significance of results, and vi) Health professional role. ConclusionLow health literacy and family conflict can complicate communication of GS results. Patients seek clear, interpretable information in a format they can easily communicate. Practice implicationsHealthcare professionals can facilitate discussion of GS results by offering written information, encouraging disclosure, exploring existing family dynamics and communication patterns, and offering strategies to improve family communication. Centralised genetic communication offices and chatbots can also be helpful

Multiple Germline Events Contribute to CancerDevelopment in Patients with Li-Fraumeni Syndrome.

Our study clarifies the genomic basis for the phenotypic variability in LFS and highlights the immense benefits of expanding genetic and epigenetic testing of patients with LFS beyond TP53. More broadly, it necessitates the dissociation of hereditary cancer syndromes as single gene disorders and emphasizes the importance of understanding these diseases in a holistic manner as opposed to through the lens of a single gene.

Strategies for the Generation of Gene Modified Avian Models: Advancement in Avian Germline Transmission, Genome Editing, and Applications.

Avian models are valuable for studies of development and reproduction and have important implications for food production. Rapid advances in genome-editing technologies have enabled the establishment of avian species as unique agricultural, industrial, disease-resistant, and pharmaceutical models. The direct introduction of genome-editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, into early embryos has been achieved in various animal taxa. However, in birds, the introduction of the CRISPR system into primordial germ cells (PGCs), a germline-competent stem cell, is considered a much more reliable approach for the development of genome-edited models. After genome editing, PGCs are transplanted into the embryo to establish germline chimera, which are crossed to produce genome-edited birds. In addition, various methods, including delivery by liposomal and viral vectors, have been employed for gene editing in vivo. Genome-edited birds have wide applications in bio-pharmaceutical production and as models for disease resistance and biological research. In conclusion, the application of the CRISPR system to avian PGCs is an efficient approach for the production of genome-edited birds and transgenic avian models.

Abstract 5954: Germline epitopes modulate immune surveillance informing breast cancer subtypes

Abstract Cancer represents a wide spectrum of molecularly and morphologically diverse diseases. Individuals with the same histopathological classification can have tumors with drastically different molecular profiles and clinical responses to treatment. It remains unclear as to when during the disease course these differences arise and why some tumors are addicted to one oncogenic pathway over another. Somatic genomic aberrations occur within the context of an individual’s germline genome, which can vary across millions of germline polymorphisms. It is unknown the extent to which germline differences influence the somatic evolution of a tumor. Interrogating 4,912 breast cancer lesions, spanning pre-invasive to metastatic disease, we demonstrate that germline variants in highly expressed and amplified genes influence somatic evolution by modulating immunoediting at early stages of tumor development. Specifically, we show that the burden of germline-derived epitopes in recurrently amplified genes selects against somatic gene amplification in pre-invasive and invasive breast cancer. As a representative example, individuals with high burden of germline-derived epitopes in ERBB2 are significantly less likely to develop HER2+ breast cancer than ER+ or triple negative breast cancer. The same negative association between epitope burden and somatic amplification is observed for four recurrent amplicons observed in high risk ER+ breast tumors. No association was observed between somatic amplifications and epitope burden in metastatic breast cancer suggesting a subset of tumors are able to overcome immune-mediated negative selection. Tumors that do are more aggressive and demonstrate an “immune cold” phenotype. Taken together, these data show the germline genome plays a pivotal role in dictating somatic evolution in breast cancer. Exploiting germline-mediated immunoediting may facilitate the development of blood-based biomarkers that refine risk stratification within breast cancer subtypes. Citation Format: Kathleen Houlahan, Aziz Khan, Christina Curtis. Germline epitopes modulate immune surveillance informing breast cancer subtypes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5954.

Abstract 6545: Investigating secondary findings in a pediatric cancer cohort: preliminary findings

Abstract Purpose: An expected outcome following germline genome sequencing in oncology is the discovery of ‘secondary findings’ (SFs). SFs comprise pathogenic(P)/likely P (LP) germline variants in cancer genes not typically associated with the presenting cancer, in addition to germline variants of uncertain significance (VUS) to the patient’s cancer. Due to the rarity of childhood cancers and a dearth of studies analyzing SFs, many pediatric SFs are categorized as VUS without clinical interpretation. Interpreting SFs poses significant challenges: VUSs and other SFs are frequently not included in clinical molecular reports, and even when reported (often through research), their clinical utility and long-term impact on patient health are unclear. However, we know VUSs can have clinical importance because some VUSs, when investigated thoroughly, have been reclassified as pathogenic predictors of significant health conditions in children. We hypothesize that an in-depth characterization of the landscape of germline SFs/VUSs across a diverse pediatric cancer cohort will reveal new roles of these genes and mutations in pediatric cancers. Methods: To explore germline SFs in pediatric cancer patients, we analyzed germline whole-genome sequencing (WGS) data for patients with rare, relapsed, refractory, and metastatic childhood cancers enrolled in the SickKids Cancer Sequencing Program (KiCS). We developed a custom analysis pipeline to identify germline single-nucleotide variants and indels deemed SFs, auto-classify their pathogenicity (ex. P, LP, or VUS) using CharGer, filter for PanCanAtlas-indicated cancer predisposition genes, and sort the remaining variants by cancer and non-cancer associations. Results: The KiCS cohort (n = 511) encompassed over 133 different tumor types; the median age of participants was 14 years (SD = 10.27) and 55% of patients were male. Ongoing work in our lab will catalogue the frequency and distribution of SFs in KiCS and analyze germline variants by subgroup (gene, tumor subtype, stage, demographics, gene function). We will also compare SF prevalence in KiCS to the general population using the gnomAD dataset. Results from preliminary analyses of this cohort will be presented. Significance: SFs/VUSs are under-utilized in cancer management. This work advances the holistic understanding of germline genomics in pediatric oncology and the roles of SFs in disease. Future studies will evaluate SFs by patient ancestry and validate cancer associations through the evaluation of allelic imbalance/loss of heterozygosity in matched tumor genomes. Citation Format: Safa Majeed, Stephenie Prokopec, Brianne Laverty, Vallijah Subasri, Michael Taylor, Yvonne Bombard, Trevor Pugh, Adam Shlien, Anita Villani, David Malkin. Investigating secondary findings in a pediatric cancer cohort: preliminary findings. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6545.

When cleaning facilitates cluttering – genome editing in ciliates

Many organisms remove DNA from their genomes during development. This has foremost been characterized as a means of defending genomes against mobile elements. However, genome editing actually hides such elements from purifying selection, with the survivors evolving approximately neutrally, ‘cluttering’ the germline genome, enabling it to enlarge over time.

Amniotes co-opt intrinsic genetic instability to protect germ-line genome integrity

Unlike PIWI-interacting RNA (piRNA) in other species that mostly target transposable elements (TEs), >80% of piRNAs in adult mammalian testes lack obvious targets. However, mammalian piRNA sequences and piRNA-producing loci evolve more rapidly than the rest of the genome for unknown reasons. Here, through comparative studies of chickens, ducks, mice, and humans, as well as long-read nanopore sequencing on diverse chicken breeds, we find that piRNA loci across amniotes experience: (1) a high local mutation rate of structural variations (SVs, mutations ≥ 50 bp in size); (2) positive selection to suppress young and actively mobilizing TEs commencing at the pachytene stage of meiosis during germ cell development; and (3) negative selection to purge deleterious SV hotspots. Our results indicate that genetic instability at pachytene piRNA loci, while producing certain pathogenic SVs, also protects genome integrity against TE mobilization by driving the formation of rapid-evolving piRNA sequences.

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.

Critical considerations for public engagement in stem cell-related research.

Public engagement is increasingly recognized as being integral to basic and translational research. Public engagement involves effective communication about research along with the mutual exchange of views and opinions among a wide variety of members in society. As such, public engagement can help to identify issues that must be addressed in order for research to be ethically sound and trustworthy. It is especially critical in research that potentially raises ethical concerns, for example research involving embryos, germline genome editing, stigmatized conditions, and marginalized communities. Therefore, it is not surprising that there have been prominent recent calls for public engagement in the emerging sciences. However, given that there is arguably little agreement about how this should be done and the best ways of doing so, those involved with planning and implementing public engagement can benefit from understanding a broad range of prior experiences on related issues.

Exploration of the Nuclear Proteomes in the Ciliate Oxytricha trifallax.

Nuclear dimorphism is a fundamental feature of ciliated protozoa, which have separate somatic and germline genomes in two distinct organelles within a single cell. The transcriptionally active somatic genome, contained within the physically larger macronucleus, is both structurally and functionally different from the silent germline genome housed in the smaller micronucleus. This difference in genome architecture is particularly exaggerated in Oxytricha trifallax, in which the somatic genome comprises tens of thousands of gene-sized nanochromosomes maintained at a high and variable ploidy, while the germline has a diploid set of megabase-scale chromosomes. To examine the compositional differences between the nuclear structures housing the genomes, we performed a proteomic survey of both types of nuclei and of macronuclear histones using quantitative mass spectrometry. We note distinct differences between the somatic and germline nuclei, with many functional proteins being highly enriched in one of the two nuclei. To validate our conclusions and the efficacy of nuclear separation, we used protein localization through a combination of transformations and immunofluorescence. We also note that the macronuclear histones strikingly display only activating marks, consistent with the conclusion that the macronucleus is the hub of transcription. These observations suggest that the compartmentalization of different genome features into separate structures has been accompanied by a similar specialization of nuclear components that maintain and facilitate the functions of the genomes specific to each nucleus.

MITE infestation accommodated by genome editing in the germline genome of the ciliate Blepharisma

During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of Blepharisma stoltei, a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.

Origins of genome-editing excisases as illuminated by the somatic genome of the ciliate Blepharisma

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout the ciliate germline genome are deleted during the development of the streamlined somatic genome. The genus Blepharisma represents one of the two high-level ciliate clades (subphylum Postciliodesmatophora) and, unusually, has dual pathways of somatic nuclear and genome development. This makes it ideal for investigating the functioning and evolution of these processes. Here we report the somatic genome assembly ofBlepharismastoltei strain ATCC 30299 (41 Mbp), arranged as numerous telomere-capped minichromosomal isoforms. This genome encodes eight PiggyBac transposase homologs no longer harbored by transposons. All appear subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We hypothesize that PiggyBac homologs were ancestral excisases that enabled the evolution of extensive natural genome editing.

N-terminal domain influences steroid activation of the Atlantic sea lamprey corticoid receptor

Lampreys are jawless fish that evolved about 550 million years ago at the base of the vertebrate line. Modern lampreys contain a corticoid receptor (CR), the common ancestor of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), which first appear in cartilaginous fish, such as sharks. Until recently, 344 amino acids at the amino terminus of adult lamprey CR were not present in the lamprey CR sequence in GenBank. A search of the recently sequenced lamprey germline genome identified two CR sequences, CR1 and CR2, containing the 344 previously un-identified amino acids. CR1 also contains a novel four amino acid insertion in the DNA-binding domain (DBD). We studied corticosteroid and progesterone activation of CR1 and CR2 and found their strongest response was to 11-deoxycorticosterone and 11-deoxycortisol, the two circulating corticosteroids in lamprey. Based on steroid specificity, both CRs are close to elephant shark MR and distant from elephant shark GR. HEK293 cells that were transfected with full-length CR1 or CR2 and the MMTV promoter have about 3-fold higher steroid-mediated activation compared to HEK293 cells transfected with these CRs and the TAT3 promoter. Deletion of the amino-terminal domain (NTD) of lamprey CR1 and CR2 to form truncated CRs decreased transcriptional activation by about 70% in HEK293 cells that were transfected with MMTV, but increased transcription by about 6-fold in cells transfected with TAT3. This indicated that the promoter has an important effect on NTD regulation of transcriptional activation of the CR by steroids. Our results also indicate that the entire lamprey CR sequence is needed for an accurate determination of steroid-mediated transcription.